Apelin fusion proteins and uses thereof

ABSTRACT

The invention provides a fusion protein or polypeptide comprising an apelin peptide fused to a multimerizing component. The invention also provides a fusion protein or polypeptide comprising an apelin peptide fused to an Fc domain, a fragment of an Fc domain, or a variant of an Fc domain. Apelin Fc-fusion polypeptides are capable of binding to the apelin receptor (APLNR). Apelin Fc-fusion polypeptides are capable of activating the APLNR and have improved pharmacokinetic properties compared to apelin peptides that are not fused to an Fc or an Fc fragment. Apelin Fc-fusion polypeptides are useful in diseases and conditions related to cardiovascular function, diabetes, cancer, obesity and other apelin-related conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC §119(e) of U.S.Provisional Patent Application No. 61/786,172, filed 14 Mar. 2013, andclaims the benefit under 35 USC §119(e) of U.S. Provisional PatentApplication No. 61/906,567, filed 20 Nov. 2013, which applications areeach specifically incorporated herein by reference in their entirety.

SEQUENCE LISTING

This application incorporates by reference the Sequence Listingsubmitted in Computer Readable Form as file 8050US_ST25.txt created onMar. 12, 2014 (43,420 bytes).

FIELD OF THE INVENTION

The present invention relates to fusion proteins engineered withmultimerizing components, such as human immunoglobulin Fc domains, fusedto the N-terminus or C-terminus of apelin peptides. Recombinant proteinsof the invention and compositions thereof, are useful in treatingcardiovascular disease, ischemia-reperfusion, diabetes, and otherapelin-related therapies.

BACKGROUND OF THE INVENTION

Preproapelin is a 77 amino acid protein expressed in the human CNS andperipheral tissues, e.g. lung, heart, and mammary gland. Peptidescomprising C-terminal fragments of varying size of apelin peptide wereshown to activate the G protein-coupled receptor, APJ receptor (Habata,et al., 1999, Biochem Biophys Acta 1452:25-35; Hosoya, et al., 2000,JBC, 275(28):21061-67; Lee, et al., 2000, J Neurochem 74:34-41;Medhurst, et al., 2003, J Neurochem 84:1162-1172). Many studies indicatethat apelin peptides and analogues convey cardiovascular actions throughtheir interaction with the APJ receptor (also known as APLNR), such asendothelium-dependent vasodilation (Tatemoto et al., 2001, Regul Pept99:87-92), positive inotropic actions (Szokodi et al., 2002, Circ Res91:434-440; Maguire, et al., 2009, Hypertension 54:598-604, epub beforeprint on Jul. 13, 2009) and myocardial regional ischemia and reperfusion(Pisarenko, et al., 2013, J Pharmacol Pharmacother. “Effects ofstructural analogues of apelin-12 in acute myocardial infarction inrats”, epub before print). Apelin-13, in particular, is a potentinotrope which could provide a treatment for heart failure by increasingheart contractility (Dai, et al., 2006, Eur J Pharmacol 553(1-3):222-228; Maguire, et al, 2009, Hypertension. 54:598-604).

Transcriptional profiling of pre- and post-surgical ventricle tissue inhuman patients revealed that APLNR was the most significantlyupregulated gene (Chen et al, 2003, Circulation, 108:1432-39). Apelin(apelin^(−/−)) and APJ (APJ^(−/−)) knockout studies in mice suggest thatlack of an endogenous apelin-APJ pathway leads to a decreased ability torespond to cardiovascular stress, such as exercise (Charo et al., 2009,Am J. Physiol. Heart Circ. Physiol., 297:H1904-1913).

Apelin has also been reported in the regulation of insulin andmechanisms of diabetes and obesity-related disorders. In mouse models ofobesity, apelin is released from adipocytes and is directly upregulatedby insulin (Boucher, et al., 2005, Endocrinol 146:1764-71). Apelinknockout mice demonstrate diminished insulin sensitivity (Yue, et al.,2010, Am J Physiol Endocrinol Metab 298:E59-E67).

APLNR-modulating agents also find utility in HIV treatment, sincesynthetic apelin peptides inhibited HIV-1 entry intoCD4-APLNR-expressing cells (Cayabyab, M., et al., 2000, J. Virol. 74:11972-11976). Furthermore, APLNR inhibitors, i.e. capable of blockingpathological angiogenesis, may be useful in inhibiting tumor growth orvascularization in the retina (Kojima, Y. and Quertermous, T., 2008,Arterioscler Thromb Vasc Biol; 28; 1687-1688; Rayalam, S. et al. 2011,Recent Pat Anticancer Drug Discov 6(3):367-72). Apelin neuroprotectionis also seen where apelin-13, apelin-17 and apelin-36 act throughsignaling pathways to promote neuronal survival (Cheng, B, et al., 2012,Peptides 37(1):171-3).

APLNR binding agents are useful in ameliorating cardiovascular disease,as well as cancer, and diabetes, among other apelin related diseases.Since apelin peptides are rapidly cleared from the circulation and havea short plasma half-life of no more than eight minutes (Japp, et al,2008, J of Amer College Cardiolog, 52(11):908-13), apelin is currentlydosed continuously to see a therapeutic effect.

There is a need in the art for improved apelin binding agents astherapeutic agents, particularly those having extended half-life, whilemaintaining APLNR binding activity.

SUMMARY OF THE INVENTION

The present invention provides apelin fusion proteins, such as apelinfused to an Fc domain, engineered to deliver biologically active apelinpeptides. In particular, apelin fusion proteins have improvedpharmacokinetic properties compared to wild-type apelin peptides whilemaintaining APLNR activity.

One aspect of the invention provides a polypeptide comprising an apelinpeptide fused to a multimerizing component. In one embodiment, themultimerizing component comprises an amino acid sequence containing atleast one cysteine residue. In another embodiment, the multimerizingcomponent comprises an amino acid sequence containing a leucine zipper,a helix-loop motif, a coiled-coil motif, or an immunoglobulin-deriveddomain. In another embodiment, the multimerizing component comprises anamino acid sequence containing an Fc domain.

In a related aspect, the invention provides a polypeptide comprising anapelin peptide fused to an Fc domain, a fragment of an Fc domain, orvariant of an Fc domain. In some cases, the polypeptide can be part of ahigher order structure, such as a protein or multimeric complex. In someembodiments, the apelin peptide is fused to the Fc domain, or fragmentthereof, via one or more peptide linkers. In other embodiments, theapelin peptide is fused to the C-terminus of said Fc domain, or theapelin peptide is fused to the N-terminus of said Fc domain, or fragmentthereof.

In one embodiment, the Fc domain of any of the apelin fusion proteinsdescribed herein comprises an immunoglobulin CH2 domain or animmunoglobulin CH3 domain. In another embodiment, the Fc domaincomprises an immunoglobulin CH2 and CH3 domain. In some embodiments, theFc domain is selected from the group consisting of IgG1 CH2 and CH3domain, IgG4 CH2 and CH3 domain, IgG1 CH2 and an IgG4 CH3 domain, andIgG4 CH2 and an IgG1 CH3 domain. In other embodiments, the Fc domaincomprises an IgG hinge domain. In still other embodiments, the Fc domaincomprises an IgG hinge domain selected from the group consisting of SEQID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, and SEQ ID NO:22.

In another embodiment, the polypeptide comprises a monomeric fusionpolypeptide capable of forming a dimer. In some embodiments, the fusionpolypeptide forms at least one disulfide bond with a second polypeptide.

In another related aspect, the invention provides an apelin receptor(APNLR) binding molecule comprising: an apelin peptide component, ahuman IgG Fc domain, and at least one linker component. In someembodiments, the apelin receptor (APNLR) binding molecule is an APLNRagonist, and in other cases the apelin receptor (APNLR) binding moleculeis an APLNR antagonist.

In another aspect of the invention, an apelin fusion polypeptide or anapelin receptor binding molecule is provided that has a plasma or serumin vivo half-life of at least about 1 hour, or at least about 2, 3, 4,5, 6, 7, 8, 9, or 10 or more hours.

In some embodiments, the apelin fusion polypeptide or receptor bindingmolecule of the invention comprises an apelin peptide selected from thegroup consisting of apelin42-77 (apelin-36), apelin61-77 (apelin-17),apelin63-77 (apelin-15), apelin64-77 (apelin-14), apelin65-77(apelin-13), apelin66-77 (apelin-12), apelin67-77 (apelin-11),apelin68-77 (apelin-10), apelin73-77 (apelin-5), apelin61-76(apelin-K16P), apelin61-75 (apelin-K15M), apelin61-74 (apelin-K14P),apelin-F13A, apelin65-76, apelin65-75, apelin66-76, apelin67-76,apelin66-75, apelin 67-75, and [Pyr¹]Apelin-13.

In certain aspects, the apelin fusion polypeptide or apelin receptorbinding molecule is a serum stable protein. In some embodiments, thepolypeptide has 95%, or 96%, or 97%, or 98%, or 99% or greater sequenceidentity to the amino acid sequence comprising SEQ ID NO: 2 or SEQ IDNO: 4. In other aspects, the polypeptide comprises an amino acidsequence at least 99% identical to SEQ ID NO: 2 or SEQ ID NO: 4. Inother aspects, the polypeptide has an amino acid sequence comprising SEQID NO: 2 or SEQ ID NO: 4. In still other aspects, the polypeptide has anamino acid sequence selected from the group consisting of SEQ ID NO: 39,SEQ ID NO: 40 and SEQ ID NO: 41.

In certain aspects, the invention provides a recombinant polypeptide,wherein the polypeptide comprises N′—P1_(m)-X1_(n)-X2-X3-P2-A1-C′,wherein: N′ is the N-terminus and C′ is the C-terminus of thepolypeptide; P1 is a peptide linker; X1 comprises an IgG hinge domain;X2 comprises an IgG CH2 domain; X3 comprises an IgG CH3 domain, P2 is apeptide linker; and A1 is an amino acid sequence comprising a humanapelin peptide, or a fragment or derivative thereof; wherein m=0 or 1,and n=0 or 1.

In certain aspects, the invention provides a recombinant polypeptide,wherein the polypeptide comprises N′-A1-P2-X1_(n)-X2-X3-C′, wherein: N′is the N-terminus and C′ is the C-terminus of the polypeptide; A1 is anamino acid sequence comprising a human apelin peptide, or a fragment orderivative thereof; P2 is a peptide linker; X1 comprises an IgG hingedomain; X2 comprises an IgG CH2 domain; and X3 comprises an IgG CH3domain; wherein n=0 or 1.

In a second aspect, the invention provides a nucleic acid moleculeencoding any apelin fusion polypeptide or apelin receptor bindingmolecule of the invention. In one embodiment, the nucleic acid moleculehas a sequence selected from the group consisting of SEQ ID NO: 27 andSEQ ID NO: 28. In other embodiments, the nucleic acid molecule encodesfor an amino acid sequence selected from the group consisting of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41.

In a third aspect, the invention provides vectors and cells comprisingthe nucleic acid molecules encoding an apelin fusion polypeptide or anapelin receptor binding molecule of the invention. In one embodiment,the vectors encode a nucleic acid molecule linked to a signal peptidesequence.

The invention also provides vectors encoding apelin fusion proteinscomprising a nucleotide sequence encoding a signal peptide. Theinvention further provides vectors encoding apelin fusion proteinscomprising a nucleotide sequence encoding a peptide linker fused to theC-terminus of a signal peptide placed upstream of the fusion protein.

In a fourth aspect, the invention provides a process for determiningAPLNR activity of a test molecule, contacting cells expressing APLNR,with the apelin fusion protein of the invention under the same testconditions as the test molecule, to determine whether the test moleculeis an APLNR agonist or an APLNR antagonist.

In one embodiment, the invention provides a process for determiningactivation of an APLN receptor (APLNR) comprising: (a) contacting cellsexpressing APLNR with a test molecule, under conditions permitting theactivation of the APLNR, (b) measuring APLNR activity, (c) separatelycontacting cells expressing APLNR with an apelin fusion protein of theinvention under the same conditions as in step (a), (d) measuring APLNRactivity of the cells in step (c) in the same manner as step (b),wherein the measurement of APLNR activity in step (b) compared to themeasurement of APLNR activity in step (d) determines that the testmolecule activates the APLNR.

Another aspect of the invention provides a method of making a fusionprotein comprising apelin, said method comprising: (a) transfecting ahost cell with a nucleic acid molecule encoding the fusion protein,wherein the nucleic acid molecule comprises a nucleotide sequenceencoding a signal peptide, fused to either i) a nucleotide sequenceencoding an Fc domain of human IgG linked to a nucleotide sequenceencoding an apelin peptide, at the N-terminus of said apelin peptide, orii) a nucleotide sequence encoding an apelin peptide linked to anucleotide sequence encoding an Fc domain of a human IgG, at theN-terminus of said Fc domain, and (b) making the fusion protein byexpressing the nucleic acid molecule of (a) in the host cell. Theinvention provides host cells secreting the fusion proteins of theinvention into the cell culture medium.

In yet another aspect, the invention provides a method for treatment ofa disease or condition related to apelin in a subject in need thereof,the method comprising administering to the subject a therapeuticallyeffective amount of the apelin fusion proteins of the invention. Theinvention also provides a method for treating the disease or conditionselected from the group consisting of cardiovascular disease, acutedecompensated heart failure, congestive heart failure, myocardialinfarction, cardiomyopathy, ischemia, ischemia/reperfusion injury,pulmonary hypertension, diabetes, obesity, cancer, metastatic disease,fluid homeostasis, pathological angiogenesis, retinopathy, fibrosis, andHIV infection, the method comprising administering to the subject atherapeutically effective amount of the apelin fusion protein of theinvention.

The invention further provides compositions and kits comprising anapelin fusion polypeptide or an apelin receptor binding molecule of theinvention. In some embodiments, the kit comprises one or more containersfilled with at least one apelin fusion protein or polypeptide of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts components of an hFc-Apelin fusion protein, such as theamino acid sequence of SEQ ID NO: 2. The sequence of SEQ ID NO: 2consists of (from N-terminus to C-terminus) human IgG1 Fc (underlined),G4S repeat peptide linker (italicized), and Apelin-13(double-underlined).

FIG. 1B represents a secreted Fc-Apelin fusion protein and itscomponents in the form of a homodimer.

FIG. 2A depicts the components of an Apelin-hFc fusion protein, such asthe amino acid sequence of SEQ ID NO: 4. The sequence of SEQ ID NO: 4consists of (from N-terminus to C-terminus) Apelin-13(double-underlined), G4S repeat peptide linker (italicized), and humanIgG1 Fc (underlined).

FIG. 2B represents a secreted Apelin-Fc fusion protein and itscomponents in the form of a homodimer.

FIG. 3A illustrates the migration of Apelin Fc-fusion proteins andprotein ladder control on an SDS-PAGE gel. Lane 1=protein markermeasurements (31 and 38 kD); Lane 2=hFc-apelin-13 (SEQ ID NO: 2); Lane3=apelin13-hFc protein (SEQ ID NO: 4); Lane 4=hFc only.

FIG. 3B illustrates the reactivity of either 10 ng or 100 ng of isolatedhFc-apelin13 or apelin13-hFc protein in a Western blot with anti-apelinantibody.

FIG. 4 represents the dose-response curve and half-maximalconcentrations (EC50s) of each of the following ligands: apelin-13(-●-), hFc-apelin13 (-▪-), or apelin13-hFc (-♦-) in a CRE-luc assay bymeasuring forskolin-induced cAMP response in APJ (APNLR)-expressingcells.

FIG. 5 represents the dose-response curve and half-maximalconcentrations (EC50s) of each of the following ligands: apelin-13(-●-), hFc-apelin13 (-▪-), apelin13-hFc (-▴-), or hFc only (-▾-) in aβ-arrestin assay.

FIG. 6 represents the normalized p-ERK assay dose-response curve andhalf-maximal concentrations (EC50s) of hFc-apelin13 (-●-) orapelin13-hFc (-▪-), compared to hFc (-▴-), showing activation ofAPNLR-expressing cells by both ligands.

FIG. 7A shows the stability of 2.8 mg/kg apelin13-hFc (-▪-) in serum ofsubcutaneously dosed C57/B16 mice, reaching levels of about 10 μg/mL forup to 48 hrs, compared to levels of hFc alone (-●-).

FIG. 7B shows stability of 5 mg/kg hFc-apelin13 (-●-) in serum ofsubcutaneously dosed C57/B16 mice, reaching 3 μg/mL at 24 hrs, andgradually decreasing to 1 μg/mL at about 14 days.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that this invention is not limited to particularmethods, and experimental conditions described, as such methods andconditions may vary. It is also to be understood that the terminologyused in this specification is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention is defined by the claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, a reference to “a method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure.

Unless defined otherwise, all technical and scientific terms used inthis application have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Although anymethods and materials similar or equivalent to those described in thisspecification can be used in the practice of the present invention,particular methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference in their entirety.

Fusion Proteins

The term “immunoglobulin” (Ig) refers to a class of structurally relatedglycoproteins consisting of two pairs of polypeptide chains, one pair oflight (L) chains and one pair of heavy (H) chains, which may all four beinter-connected by disulfide bonds. The structure of immunoglobulins hasbeen well characterized. See for instance Fundamental Immunology Ch. 7(Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Each heavy chaintypically comprises a heavy chain variable region (abbreviated herein asV_(H) or VH) and a heavy chain constant region (C_(H) or CH). The heavychain constant region typically comprises three domains, CH1, CH2, andCH3. The CH1 and CH2 domains are linked by a hinge. The Fc portioncomprises at least the CH2 and CH3 domains.

Typically, the numbering of amino acid residues of immunoglobulins isaccording to IMGT, Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md.(1991), or by the EU numbering system of Kabat (also known as “EUnumbering” or “EU index”), e.g., as in Kabat, E. A. et al. Sequences ofProteins of Immunological interest. 5^(th) ed. US Department of Healthand Human Services, NIH publication No. 91-3242 (1991).

As used in the specification, a “multimerizing component” is anymacromolecule, protein, polypeptide, peptide, or amino acid that has theability to associate with a second multimerizing component of the sameor similar structure or constitution. For example, a multimerizingcomponent may be a polypeptide comprising an immunoglobulin C_(H)3domain. A non-limiting example of a multimerizing component is an Fcportion of an immunoglobulin, e.g., an Fc domain of an IgG selected fromthe isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype withineach isotype group. In certain embodiments, the multimerizing componentis an Fc fragment or an amino acid sequence of 1 to about 500 aminoacids in length containing at least one cysteine residues. In otherembodiments, the multimerizing component is a cysteine residue, or ashort cysteine-containing peptide. Other multimerizing domains includepeptides or polypeptides comprising or consisting of a leucine zipper, ahelix-loop motif, or a coiled-coil motif.

The term “Fc” refers to a portion of a heavy chain constant region thatcomprises at least the CH2 and CH3 domains that typically bind to an Fcreceptor e.g., an FcγR, namely FcγRI (CD64), FcγRII (CD32), FcγRIII(CD16) or an FcRn, i.e., a neonatal Fc receptor. If the CH2 and CH3region contains deletions, substitutions, and/or insertions or othermodifications that render it unable to bind any Fc receptor, then theCH2 and CH3 region is considered to be non-functional in terms of itstypical biological function.

The phrase “fusion proteins”, and specifically “apelin fusion proteins”,includes recombinant polypeptides and proteins derived from apelin thathave been engineered to contain a multimerizing component as describedherein.

The phrase “Fc-fusion proteins”, and specifically “apelin-Fc” or“Fc-apelin” fusion proteins, includes recombinant polypeptides andproteins derived from apelin that have been engineered to contain an Fcfragment as described herein. For example, an “apelin Fc-fusion protein”includes a chimeric protein comprising an amino acid sequence of anapelin peptide or analogue fused to an amino acid sequence of an Fcdomain of Ig, either at the N-terminus or the C-terminus, with orwithout peptide linkers. Examples of peptides used in fusion proteinsare known in the art (see e.g. Dumont, et al., 2006, Biodrugs20(3):150-160). Fc-fusion proteins are also referred to in the art asimmunoadhesins.

The phrase “fused to”, as used herein, means (but is not limited to) apolypeptide formed by expression of a chimeric gene made by combiningmore than one sequence, typically by cloning one gene into an expressionvector in frame with a second gene such that the two genes are encodingone continuous polypeptide. In addition to being made by recombinanttechnology, parts of a polypeptide can be “fused to” each other by meansof chemical reaction, or other means known in the art for making custompolypeptides.

The term “protein” is meant to include quaternary structures, ternarystructures and other complex macromolecules composed of at least onepolypeptide. The term “protein” includes polypeptide.

As used herein, a “polypeptide” is a single linear polymer chain ofamino acids bonded together by peptide bonds between the carboxyl andamino groups of adjacent amino acid residues. The term “protein” mayalso be used to describe a large polypeptide, such as a seventransmembrane spanning domain protein.

The polypeptides of the invention comprise amino acid sequences that arederived from an immunoglobulin domain. A polypeptide or amino acidsequence “derived from” a designated protein or polypeptide refers tothe origin of the polypeptide. As used herein, “isotype” refers to theimmunoglobulin class or subclass (for instance, IgG1, IgG2, IgG3, IgG4,IgD, IgA, IgE, or IgM) that is encoded by heavy chain constant regiongenes.

The phrase “heavy chain” or “immunoglobulin (Ig) heavy chain”, as usedherein, includes Ig heavy chain constant region sequence from anyorganism, and unless otherwise specified includes a heavy chain variabledomain. Heavy chain variable domains include three heavy chaincomplementary determining regions (CDRs) and four framework regions(FRs), unless otherwise specified. Fragments of heavy chain variabledomains include CDRs, or both CDRs and FRs. A typical heavy chainconstant region (CH) has, following the variable domain, from N-terminalto C-terminal: a CH1 domain, a hinge, a CH2 domain, and a CH3 domain. Afunctional fragment of a heavy chain, e.g. in an antigen-bindingprotein, includes a fragment that is capable of specifically recognizingan antigen (e.g., recognizing the antigen with a K_(D) in themicromolar, nanomolar, or picomolar range), that is capable of beingexpressed in and secreted from a cell, and that comprises at least oneCDR.

Flow cytometry-based autologous secretion trap (FASTR) methods, whichutilize a membrane-bound human Fey receptor (hFcγR) to captureco-secreted proteins, can be used to rapidly isolate high expressionclones expressing or secreting an antibody or Fc-fusion protein. (See,US20090137416 A1, which is herein incorporated by reference.) Such highexpression clones may be employed to isolate cells expressing proteinscomprising an Fc-fusion protein as described herein. FASTR methods maybe utilized to directly screen and isolate cells expressing anyrecombinant polypeptide or Fc-fusion protein of the invention.

The term “hinge”, as used herein, is intended to include the region ofconsecutive amino acid residues that connect the C-terminus of the CH1to the N-terminus of the CH2 domain of an immunoglobulin. Several aminoacids of the N-terminus of the CH2 domain, which are coded by the CH2exon, are also considered part of the “lower hinge”. Without being boundby any one theory, amino acids of the hinge region of IgG1, IgG2 andIgG4 have been characterized as comprising 12-15 consecutive amino acidsencoded by a distinct hinge exon, and several N-terminal amino acids ofthe CH2 domain (encoded by the CH2 exon) (Brekke, O. H., et al., 1995,Immunology Today 16(2):85-90). On the other hand, IgG3 comprises a hingeregion consisting of four segments: one upper segment resembling thehinge region of IgG1, and 3 segments that are identical amino acidrepeats unique to IgG3.

Amino acid residues derived from Ig domains, such as human IgG, areidentified herein by the EU numbering system of Kabat, also known as “EUnumbering” or the “EU index” (according to Kabat, E. A. et al. Sequencesof Proteins of Immunological interest. 5^(th) ed. US Department ofHealth and Human Services, NIH publication No. 91-3242, 1991, andupdated according to the IMGT® Scientific Chart, IMGT, the internationalImMunoGeneTics information System®,http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html,created: 17 May 2001, last updated: 10 Jan. 2013).

For example, EU numbering for human IgG1 hinge amino acids and thecorresponding IMGT unique numbering convention, and the Kabat numberingconvention (according to Kabat, E. A. et al, 1991, and IMGT® ScientificChart supra) are listed in Table 1.

TABLE 1 IgG1 hinge numbering IgG1 (IGHG1) IMGT Unique amino acidsNumbering for Kabat [SwissProt P01857] the hinge EU Numbering Numbering(E) 1 216 226 P 2 217 227 K 3 218 228 S 4 219 232^(a) [229]^(b) C 5 220233^(a) [230]^(b) D 6 221 234^(a) [232]^(b) K 7 222 235 T 8 223 236 H 9224 237 T 10 225 238 C 11 226 239 P 12 227 240 P 13 228 241 C 14 229 242P 15 230 243 Amino acids resulting from exon splicing are shown inparentheses. ^(a)numbering according to the last updated IMGT ScientificChart ^(b)numbering according to EU index as originally reported inKabat, EA, et al. 1991 See also, e.g., Lefranc, M.-P. et al., Devel CompImmunol, 29, 185-203 (2005); and Edelman, G. M. et al. PNAS USA, 63:78-85 (1969).

TABLE 2 IgG1 C-domain hinge numbering IgG1 (IGHG1) IMGT Unique aminoacids Numbering for Kabat [SwissProt P01857] C-domains EU NumberingNumbering (A) 1.6 231 244 P 1.5 232 245 E 1.4 233 246 L 1.3 234 247 L1.2 235 248 G 1.1 236 249

In one embodiment, Fc-fusion proteins of the invention comprise an Fcdomain or any Fc domain fragment or any Fc domain variant. In someembodiments, the Fc domain comprises an Ig CH2 and an Ig CH3 domain, ora fragment or variant thereof. In other embodiments, the Fc domaincomprises an Ig hinge domain, or a fragment or variant thereof, an IgCH2 domain or a fragment or variant thereof and an Ig CH3 domain or afragment or variant thereof. In still other embodiments, the Fc domaincomprises an Ig CH1 domain or a fragment or variant thereof, an Ig hingedomain or a fragment or variant thereof, an Ig CH2 domain a fragment orvariant thereof, and an Ig CH3 domain a fragment or variant thereof.

The term “chimeric”, as used herein, means composed of parts ofdifferent origin. The phrase “chimeric protein”, which encompasses“chimeric polypeptides”, includes a first amino acid polypeptide linkedto a second amino acid polypeptide that is not normally linked innature. The amino acid sequences may normally exist as separatepolypeptides or in a different arrangement on the same polypeptide orprotein, and are brought together in a fusion polypeptide in a newarrangement.

The Fc domain may be chimeric, combining Fc sequences derived from morethan one immunoglobulin isotype. For example, a chimeric Fc domain cancomprise part or all of a CH2 sequence derived from a human IgG1, humanIgG2 or human IgG4 CH2 region, and part or all of a CH3 sequence derivedfrom a human IgG1, human IgG2 or human IgG4. A chimeric Fc domain canalso contain a chimeric hinge region. For example, a chimeric hinge maycomprise an “upper hinge” sequence, derived from a human IgG1, a humanIgG2 or a human IgG4 hinge region, combined with a “lower hinge”sequence, derived from a human IgG1, a human IgG2 or a human IgG4 hingeregion. A chimeric Fc domain can have altered Fc receptor binding, whichin turn affects Fc effector function.

For certain therapies, the Fc domain may be engineered to activate all,some, or none of the normal Fc effector functions, without affecting thedesired Fc-fusion protein's pharmacokinetic properties. Therefore,engineered Fc domains that have altered Fc receptor binding may havereduced side effects. Thus, in one embodiment, the protein comprises achimeric or otherwise modified Fc domain. For an example of a chimericFc domain, see U.S. Provisional Application No. 61/759,578, filed Feb.1, 2013, which is herein incorporated in its entirety.

The invention also provides apelin Fc-fusion proteins comprising variantFc domain sequences. Such “variant” Fc domains and Fc domain fragmentscomprise one or more additions, deletions, or substitutions of aminoacids when compared to wild-type sequence, but essentially function asdesired, e.g. exhibit APLNR activity and prolong half-life of the fusionprotein, as described in this specification.

In some embodiments, the Fc domain comprises an IgG CH2 and CH3 domain.In other embodiments, the Fc domain comprises an IgG1 CH2 and CH3domain, IgG4 CH2 and CH3 domain, IgG1 CH2 domain and an IgG4 CH3 domain,or IgG4 CH2 domain and an IgG1 CH3 domain. In some embodiments, the Fcdomain is a chimeric Fc domain comprising a fragment selected from thegroup consisting of CH1 domain, hinge domain, CH2 domain and CH3 domain,wherein the fragment is derived from IgG1, IgG2, IgG3, IgG4, IgD, IgA,IgE, or IgM. In some embodiments, the chimeric Fc domain comprises a CH2domain selected from the group consisting of SEQ ID NO: 15, SEQ ID NO:19, and SEQ ID NO: 23. In some embodiments, the chimeric Fc domaincomprises a CH3 domain selected from the group consisting of SEQ ID NO:16, SEQ ID NO: 20, and SEQ ID NO: 24. In another embodiment, the Fcdomain comprises a chimeric IgG CH2-CH3 domain. Accordingly, variantsand fragments of such Fc domains are also part of this invention.

In one embodiment, the Fc domain comprises an IgG1, IgG2, IgG3 or IgG4hinge domain. In one embodiment, the hinge domain comprises SEQ ID NO:14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21 or SEQ ID NO: 22. Inanother embodiment, the Fc domain comprises a chimeric hinge domain. Inanother embodiment, the Fc domain comprises a chimeric hinge domaincomprising a hinge fragment selected from the group consisting of SEQ IDNO: 14, SEQ ID NO: 18, and SEQ ID NO: 22.

According to certain embodiments of the present invention, Fc-fusionproteins are provided comprising an Fc domain comprising one or moremutations which enhance or diminish protein binding to the FcRnreceptor, e.g., at acidic pH as compared to neutral pH. For example, thepresent invention includes Fc-fusion proteins comprising a mutation inthe CH2 or a CH3 region of the Fc domain, wherein the mutation(s)increases the affinity of the Fc domain to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Suchmutations may result in an increase in serum half-life of the antibodywhen administered to an animal. Non-limiting examples of such Fcmodifications include, e.g., a modification at position 250 (e.g., E orQ); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., Sor T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or amodification at position 250 and/or 428; or a modification at position307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, themodification comprises a 428L (e.g., M428L) and 434S (e.g., N434S)modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F)modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification;a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Qand 428L modification (e.g., T250Q and M428L); and a 307 and/or 308modification (e.g., 308F or 308P).

For example, the present invention includes Fc-fusion proteinscomprising an Fc domain comprising one or more pairs or groups ofmutations selected from the group consisting of: 250Q and 248L (e.g.,T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E);428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433Kand N434F). All possible combinations of the foregoing Fc domainmutations, and other mutations within the fusion proteins disclosedherein, are contemplated within the scope of the present invention.

Modifications to the Fc domain of an Fc-fusion protein may conferincreased stability, such as resistance to degradation. Fusion proteinsmay be modified using ordinary molecular biological techniques andsynthetic chemistry so as to improve their resistance to proteolyticcleavage or resistance to metal ion-related cleavage. Analogues of suchpolypeptides include substitution variants made by the exchange of oneamino acid for another or substitution with residues other thannaturally occurring L-amino acids, e.g. D-amino acids or non-naturallyoccurring synthetic amino acids.

In one embodiment, Fc-fusion proteins of the invention comprise anapelin peptide fused to an Fc domain as described herein.

In some embodiments, the Fc-fusion protein of the invention activatesthe APLNR receptor and has a greater half-life than that of an apelinpeptide that is not fused to an Fc domain, such as a greater half-lifeof more than eight minutes.

Apelin Ligand and Apelin Receptor

Apelin is produced endogenously as a prepropeptide of 77 amino acidswhich is cleaved to yield several shorter biologically active fragments,or apelin peptides.

In some embodiments, Fc-fusion proteins of the invention comprise anapelin peptide as described herein.

In some embodiments, the apelin peptide comprises a fragment orderivative of the preproapelin polypeptide (SEQ ID NO: 5).

“Apelin peptides” includes specific apelin fragments and derivativesknown in the art, e.g., an apelin peptide comprising amino acids 6-77,40-77, 42-77, 43-77, 47-77, 59-77, 61-77, 63-77, 64-77, 65-77, 66-77,67-77, 73-77, 1-25, 6-25, 42-64, 61-64, 61-74, 61-75, 61-76, 65-76,65-75, 66-76, 67-76, 66-75, 67-75, 42-58, 42-57, 42-56, 42-55, 42-54,42-53, or pyroglutamylated apelin65-77 ([Pyr¹]Apelin-13), of thepreproapelin polypeptide (SEQ ID NO: 5). See e.g. U.S. Pat. No.6,492,324, issued on Dec. 10, 2002, and El Messari et al. 2004, JNeurochem, 90:1290-1301, which are both herein incorporated byreference. In one embodiment, the apelin peptide comprises amino acids65-76, 65-75, 61-77, 63-77, 64-77, 65-77, 66-77, 67-77, 66-76, 67-76,66-75, 67-75, or 42-77 of SEQ ID NO: 5.

It has been demonstrated herein that fragments of apelin peptides, forexample peptides having C-terminal deletions, retain their cellularactivities (see also El Messari et al. 2004, J Neurochem, 90:1290-1301).Certain apelin peptide derivatives, such as apelin peptides and fusionshaving additional one or more C-terminal amino acids, are shown hereinto retain their cellular activities. As such, fragments and derivativesof the apelin peptides described in this specification are included inthe invention. Other fragments and derivatives of apelin peptides may bemade by recombinant technology by the skilled artisan.

In other embodiments, the apelin peptide is selected from the groupconsisting of apelin40-77 (apelin-38), apelin42-77 (apelin-36),apelin43-77 (apelin-35), apelin47-77 (apelin-31), apelin59-77(apelin-19), apelin61-77 (apelin-17), apelin63-77 (apelin-15),apelin64-77 (apelin-14), apelin65-77 (apelin-13), apelin66-77(apelin-12, or A12), apelin67-77 (apelin-11), apelin68-77 (apelin-10),apelin73-77 (apelin-5), apelin61-76 (apelin-K16P), apelin61-75(apelin-K15M), apelin61-74 (apelin-K14P), and [Pyr¹]Apelin-13.

In still other embodiments, the apelin peptide is selected from thegroup consisting of apelin61-77 (apelin-17; SEQ ID NO: 7), apelin65-77(apelin-13; SEQ ID NO: 6), apelin-F13A (SEQ ID NO: 29), apelin66-77(apelin-12, or A12, SEQ ID NO: 32), apelin67-77 (apelin-11; SEQ ID NO:33), apelin65-76 (SEQ ID NO:30), apelin65-75 SEQ ID NO: 31), apelin67-77(SEQ ID NO: 6), apelin66-76 (SEQ ID NO: 34), apelin67-76 (SEQ ID NO:35), apelin 66-75 (SEQ ID NO: 36), apelin 67-75 (SEQ ID NO: 37), and[Pyr¹]Apelin-13.

In some embodiments, the apelin peptide is modified to minimizedegradation and to enhance serum stability. In certain embodiments, themodified apelin peptide is selected from the group consisting of SEQ IDNO: 38 (apelin-13+5G), SEQ ID NO: 42 (apelin-13+R), SEQ ID NO: 43(apelin-13+S), and SEQ ID NO: 44 (apelin-13+H).

In one embodiment, the apelin peptide is selected from the groupconsisting of apelin-36 (SEQ ID NO: 8), apelin-17 (SEQ ID NO: 7),apelin-13 (SEQ ID NO: 6) and [Pyr¹]Apelin-13. In another embodiment, theapelin peptide comprises apelin-13 (SEQ ID NO: 6), or a fragmentthereof.

Apelin peptides are rapidly cleared from the circulation and have ashort plasma half-life of no more than eight minutes (Japp, et al, 2008,J of Amer College Cardiolog, 52(11):908-13). Apelin fusion proteins ofthe invention have increased half-life compared to apelin peptides.

Included in the invention are analogues of apelin modified to includenon-standard amino acids or modified amino acids. Such peptidescontaining non-natural, or natural but non-coded, amino acids may besynthesized by an artificially modified genetic code in which one ormode codons is assigned to encode an amino acid which is not one of thestandard amino acids. For example, the genetic code encodes 20 standardamino acids, however, three additional proteinogenic amino acids occurin nature under particular circumstances: selenocysteine, pyrrolysineand N-Formyl-methionine (Ambrogelly, et al. 2007, Nature ChemicalBiology, 3:29-35; Böck, A. et al, 1991, TIBS, 16 (12): 463-467; andThéobald-Dietrich, A., et al., 2005, Biochimie, 87(9-10):813-817).Post-translationally modified amino acids, such as carboxyglutamic acid(γ-carboxyglutamate), hydroxyproline, and hypusine, are also included.Other non-standard amino acids include, but are not limited to,citrulline, 4-benzoylphenylalanine, aminobenzoic acid, aminohexanoicacid, N^(α)-methylarginine, α-Amino-n-butyric acid, norvaline,norleucine, alloisoleucine, t-leucine, α-Amino-n-heptanoic acid,pipecolic acid, α,β-diaminopropionic acid, α,γ-diaminobutyric acid,ornithine, allothreonine, homoalanine, homoarginine, homoasparagine,homoaspartic acid, homocysteine, homoglutamic acid, homoglutamine,homoisoleucine, homoleucine, homomethionine, homophenylalanine,homoserine, homotyrosine, homovaline, isonipecotic acid, β-Alanine,β-Amino-n-butyric acid, β-Aminoisobutyric acid, γ-Aminobutyric acid,α-aminoisobutyric acid, isovaline, sarcosine, naphthylalanine, nipecoticacid, N-ethyl glycine, N-propyl glycine, N-isopropyl glycine, N-methylalanine, N-ethyl alanine, N-methyl β-alanine, N-ethyl β-alanine,octahydroindole-2-carboxylic acid, penicillamine, pyroglutamic acid,sarcosine, t-butylglycine, tetrahydro-isoquinoline-3-carboxylic acid,isoserine, and α-hydroxy-γ-aminobutyric acid. A variety of formats toexpand the genetic code are known in the art and may be employed in thepractice of the invention. (See e.g. Wolfson, W., 2006, Chem Biol,13(10): 1011-12.)

Apelin analogues incorporating such non-standard amino acids orpost-translational modifications can be synthesized by known methods.Exemplary apelin analogues include N^(α)-methylarginine-apelin-A12analogue, [Nle⁷⁵, Tyr]apelin-36, [Glp⁶⁵Nle⁷⁵, Tyr⁷⁷]apelin-13,(Pyr¹)[Met(O)11]-apelin-13, (Pyr¹)-apelin-13, [d-Ala¹²]-A12, andN-alpha-acetyl-nona-D-arginine amide acetate.

Also included in the invention are analogues of the apelin component ofan apelin fusion protein modified to be resistant to cleavage, forexample cleavage by angiotensin converting enzyme 2 (ACE2). Such apelinanalogues have been shown to have a marked increase in efficacy comparedto unmodified apelin ligands in in vivo models of myocardial response toischemia (Wang, et al. Jul. 1, 2013, J Am Heart Assoc. 2: e000249).

Such cleavage-protected apelin fusion proteins comprise apelin peptidesthat are modified to include substitution variants, i.e. variants madeby the exchange of one amino acid for another at one or more cleavagesites within the protein. Such amino acid substitutions are envisionedto confer increased stability without the loss of other functions orproperties of the protein. Other cleavage-protected apelin fusionproteins comprise apelin peptides modified to include terminal amide oracetyl groups. In some embodiments, cleavage-protected apelin fusionproteins comprise proteinogenic amino acids, non-standard amino acids orpost-translationally modified amino acids. Still othercleavage-protected or cleavage-resistant apelin fusion proteins comprisemodified apelin peptides that include one or more additional N-terminalamino acids. It is desirable that such modified apelin peptides do notalter the peptide's ability to activate the APLNR. Exemplary modifiedapelin peptides and fusion proteins of the invention that activate APLNRinclude SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44.

Apelin, as mentioned above, is known to be a ligand of APLNR, a Gprotein-coupled receptor. The term “ligand”, as used herein, means amolecule that binds to another molecule such as a receptor. A ligandmolecule capable of binding to a G protein-coupled receptor (GPCR) isselected from the group consisting of an ion, small organic molecule,peptide, polypeptide, antibody, bispecific antibody, antibody fragment,protein, and large organic molecule. A ligand may be furthercharacterized as, for example, an agonist, partial agonist, inverseagonist, antagonist, competitive antagonist, positive allostericmodulator or negative allosteric modulator depending on the state ofactivity it confers through the receptor to which it binds. For example,for agonists to bind to a GPCR, other molecular interactions that keepsuch GPCR in an inactive state are disrupted.

The term “agonist”, as used herein, includes a moiety that interactswith (directly or indirectly binds) and activates a receptor, such asthe APLNR receptor, and initiates a physiological or pharmacologicalresponse characteristic of that receptor, such as when bound to itsendogenous ligand. For example, upon binding to a GPCR, moieties mayactivate an intracellular response, enhance GTP binding to cellmembranes, or internalize the receptor. Such agonist moiety can be forexample a protein, polypeptide, peptide, antibody, antibody fragment,large molecule, or small molecule.

The term “antagonist”, as used herein, is intended to mean a moiety thatcompetitively binds to the receptor at the same site as an agonist (forexample, the endogenous ligand), but which does not activate theintracellular response initiated by the active form of the receptor, andthereby inhibits the intracellular response by an agonist or partialagonist. In some cases, antagonists do not diminish the baselineintracellular response in the absence of an agonist or partial agonist.An antagonist does not necessarily have to function as a competitivebinding inhibitor, but may work by sequestering an agonist, orindirectly modulating a downstream effect.

G protein-coupled receptors (GPCRs), which are seven transmembranedomain receptors, typically transduce their cellular signals viaheterotrimeric guanine nucleotide-binding proteins (G proteins),consisting of an alpha (α), beta (β), and gamma (γ) subunit, whereas theα subunit contains a binding site for GTP/GDP, and the βγ dimer is boundto the α subunit in an inactive state. G proteins are naturallyoccurring on the cytoplasmic side of the plasma membrane. Binding of anextracellular ligand leads to a conformational change in the receptorprotein that allows it to make contact with a guanine-nucleotide bindingprotein (G protein), and thus enhance the exchange of GTP for GDP. Uponthe exchange, the βγ dimer dissociates from the α subunit. Both theactivated α subunit and the βγ dimer can influence intracellulareffector proteins.

In general, GPCRs activate a particular Gα protein subunit family, whichleads to the activation or inactivation of a particular signaltransduction pathway. The apelin receptor (APLNR) is a GPCR.

Upon interaction with a ligand or binding molecule, the apelin receptor(APLNR) triggers one or more of several intracellular signaling cascadesincluding signaling initiated by: 1) inhibitory G protein subunit,Gα_(i/o), 2) activation of ERKs through PKC, or 3) internalization ofthe GPCR. In other words, the pharmacological and/or physiologicalresponse of APLNR in its active state is determined by the downstreamaction of Gα_(i) subunits (which, e.g., inhibit adenylyl cyclase),phosphorylated ERKs or internalized APLNR. It is understood that otherintracellular effectors may be engaged by an activated APLNR.

Apelin receptor (APLNR) originally named APJ receptor (O'Dowd, et al.,1993, Gene 136(1-2):355-360), was isolated from human genomic DNA as a380 amino acid 7-transmembrane domain orphan receptor. (See NCBI RefSeqNo. NP_005152, which is herein incorporated by reference.) Apelin wasshown to be the endogenous ligand for APLNR (APJ) when tissue extractsfrom bovine stomach revealed apelin peptides that stimulatedacidification rate in CHO cells expressing APLNR (APJ) in a range from0.1-100 nM (Tatemoto, et al., 1998, Biochem Biophys Res Comm251:471-476).

The interaction between apelin and APLNR, and hence the interactionbetween apelin fusion proteins and APLNR, can be measured by a number ofin vitro (e.g. as in a test tube or plate), ex vivo (e.g. as in a cellculture from a living animal) and in vivo (e.g. as in a living animal)bioassays known to the skilled person in the relevant art.

In some embodiments, APLNR agonists are selected from the groupconsisting of apelin-36, apelin-19, apelin-17, apelin-13, apelin-12,N^(α)-methylarginine-apelin-A12 analogue, [Nle⁷⁵, Tyr]apelin-36,[Glp⁶⁵Nle⁷⁵, Tyr⁷⁷]apelin-13, (Pyr¹)[Met(O)11]-apelin-13, and(Pyr¹)-apelin-13.

In one embodiment, the apelin fusion polypeptide is an APLNR agonistselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, and fragments or derivativesthereof.

Antagonists of the receptor are known to block the hypotensive action ofthe APLNR. The apelin peptide derivative made by modifying apelin-13 atits C-terminal phenylalanine (F) to alanine (A) (apelin-13(F13A); SEQ IDNO: 29) was described by Lee, et al. 2005 (Endocrinol 146(1):231-236) asa functional antagonist. The APLNR antagonist F13A was also reported toimprove circulatory and renal function in cirrhotic animals, indicatingthat the antagonist may have mediated the overactive effects of anupregulated apelin system in pathogenic disease such as fibrosis of theliver (Principe, A., et al. 2008, Hepatology, 48(4):1193-1201). In someembodiments, APLNR antagonists are selected from the group consisting ofapelin-13(F13A) (SEQ ID NO: 29), [d-Ala¹²]-A12,cyclo(1-6)CRPRLC-KH-cyclo(9-14)CRPRLC, andN-alpha-acetyl-nona-D-arginine amide acetate (ALX40-4C; CAS Registry No.153127-49-2).

In another embodiment, the apelin fusion protein or polypeptidecomprises an APLNR binding molecule. In other embodiments, the apelinfusion protein or polypeptide comprises an APLNR agonist. In someembodiments, the fusion polypeptide of the invention comprises an APLNRantagonist.

Receptor Assays

It is understood that receptor screening assays are employed not only tothe subject apelin fusion proteins of the invention, but also any testcompounds including agonists and antagonists of the APLNR. Many receptorscreening assays to determine activation or inactivation of the APLNRare well-known in the art, and the following examples are not intendedto limit the scope of what the inventors regard as their invention.

GPCR-mediated guanine nucleotide exchange is monitored by measuring[³⁵S]GTPγS binding to plasma membranes prepared from cells expressingGPCRs of interest. The [³⁵S]GTPγS assay is generally useful forGi/o-coupled receptors because Gi/o is the most abundant G protein inmost cells and has a faster GDP-GTP exchange rate than other G proteins(Milligan G., 2003, Trends Pharmacol Sci, 2003, 24:87-90). Commerciallyavailable Scintillation Proximity Assay (SPA™) kits allow measurement ofdesired [³⁵S]GTPγS-bound α subunit (Perkin Elmer, Waltham, Mass., USA).

Activation of Gi/o-coupled receptors results in decreased adenylylcyclase activity and therefore inhibition of cAMP in the cell, via the Galpha subunits Gi or Go. To maximize the inhibition signal, forskolin (adirect activator of adenylate cyclase) is typically utilized tostimulate adenylyl cyclase in the assay, and thus cAMP, therebyrendering the inhibition signal more easily detectable. Radiometric GEHealthcare SPA™ (Piscataway, N.J., USA) and Perkin Elmer Flash-Plate™cAMP assays are available, as well as fluorescence or luminescence-basedhomogenous assays (e.g. PerkinElmer AlphaScreen™, DiscoveRx HitHunter™(Fremont, Calif., USA), and Molecular Devices FLIPR® (Sunnyvale, Calif.,USA)) to measure accumulation of intracellular cAMP.

The action of GPCRs that modulate cAMP levels, like APLNR, may be linkedto luciferase transcription in a cell by a cAMP response element (CRE).A CRE-luc construct (CRE-responsive luciferase) encodes a luciferasereporter gene under the control of a promoter and tandem repeats of theCRE transcriptional response element (TRE). Following activation of thereceptor, cAMP accumulation in the cell is measured by the amount ofluciferase expressed in the cell following addition of chemiluminescentdetection reagents. For APLNR, and other Gi-coupled receptors, forskolinis added to induce cAMP and a decrease in CRE activity(chemiluminescence) indicates GPCR activation. Various commercial kitsare available, such as from Promega (Madison, Wis., USA), SABiosciences(A Qiagen Company, Valencia, Calif., USA), etc.

In some instances, agonist binding to the receptor may initiatearrestin-mediated signaling, without triggering G protein-mediatedsignaling or slow down G protein-mediated signaling. Beta-arrestin(β-arrestin) interaction with GPCRs at the cell-surface can uncoupleheterotrimeric G proteins to the receptor and lead to other cellsignaling cascades. β-arrestin is known to trigger endocytosis andactivation of the ERK pathway. In one example assay, bioluminescenceresonance energy transfer or BRET has been used to study the interactionof GPCRs fused to Renilla luciferase (Rlu) with β-arrestin fused togreen fluorescent protein (GFP). In this example, BRET is based on thetransfer of energy between recombinant expressed GPCR-Rlu andβ-arrestin-GFP when they are in close proximity after the addition ofthe luciferase substrate coelentcrazine, thus allowing measurement ofreal-time evaluation of these protein-protein interactions in wholecells.

Other assays have been developed, such as PathHunter® GPCR assays(DiscoveRx Corp., Fremont, Calif., USA) that directly measure GPCRactivity by detecting β-arrestin interaction with the activated GPCR.Briefly, the GPCR is fused in frame with the small enzyme fragmentProLink™ and co-expressed in cells stably expressing a fusion protein ofβ-arrestin and a deletion mutant of β-galactosidase (i.e. β-gal, anenzyme acceptor, or EA). Activation of the GPCR stimulates binding ofβ-arrestin to the ProLink-tagged GPCR and the complementation of the twoenzyme fragments results in formation of an active β-gal enzyme. Anincrease in enzyme activity (i.e. GPCR activation) can be measured usingchemiluminescent detection reagents.

β-arrestin molecules have been shown to regulate GPCR internalization(i.e. endocytosis) following activation of GPCRs, such as APLNR.Agonist-activation of GPCRs leads to conformational changes,phosphorylation of the receptor, and activation of β-arrestin, or otherpathways, to mediate receptor sequestration from the cell surface. Thesequestration mechanism may be a means of desensitization (i.e. receptoris degraded following internalization) or resensitization (i.e. receptoris recycled back to the cell surface). See, e.g., Claing, A., et al.2002, Progress in Neurobiology 66: 61-79, for review.

APLNR antagonists may block internalization of the receptor. APLNRagonists may induce internalization and/or resensitization of the APLNR(Lee, D K, et al. 2010, BBRC, 395:185-189). In some embodiments, theAPLNR agonist exhibits or induces increased APLNR resensitization, asmeasured by an internalization assay. In other embodiments, the APLNRagonist exhibits or induces increased cell-surface receptor copy of theAPLNR, as measured in an internalization assay. Measuring the extent(such as an increase) of receptor internalization in any internalizationassay is done by determining the difference between the noninternalizedmeasurement (i.e., cells without prior exposure to agonist) and themeasurement obtained with agonist in the assay.

Apelin receptor sequestration, and thus apelin receptor copy, may bemeasured by a number of methods well-known in the art. APLNR agoniststimulation may result in increased or decreased receptor copy on thesurface of a particular cell. For example, an apelin receptor agonistthat induces APLNR internalization may have an effect on blood pressure.Receptor internalization assays are routinely done employing, forexample, fluorescently-labeled or radiolabeled ligands, orimmunofluorescent labels (fluorescently-tagged anti-receptorantibodies), followed by microscopy and digital imaging techniques (see,e.g., El Messari et al. 2004, J Neurochem, 90:1290-1301; and Evans, N.,2004, Methods of Measuring Internalization of G Protein-CoupledReceptors. Current Protocols in Pharmacology. 24: 12.6.1-12.6.22).

Phosphorylated ERK (p-ERK) may be measured in cell lysates from cellsexpressing APLNR receptors to determine APLNR activation. Endogenousextracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2), belong toa conserved family of serine/threonine protein kinases and are involvedcellular signaling events associated with a range of stimuli. The kinaseactivity of ERK proteins is regulated by dual phosphorylation atThreonine 202/Tyrosine 204 in ERK1, and Threonine 185/Tyrosine 187 inERK2. MEK1 and MEK2 are the primary upstream kinases responsible for ERK1/2 in this pathway. Many downstream targets of ERK 1/2 have beenidentified, including other kinases, and transcription factors. In oneexample, the p-ERK 1/2 assay utilizes an enzyme-linked immunosorbentassay (ELISA) method to measure specific phosphorylation of ERK 1 incellular lysates of cell cultures expressing recombinant or endogenousreceptors. In another example, the p-ERK 1/2 assay uses a primary(non-conjugated) antibody which recognizes phosphorylated Thr202/Tyr204in ERK1 or phos-Thr185/Tyr187 in ERK2 and a secondary conjugatedantibody that recognizes the primary antibody, whereas the secondaryconjugated mAb provides a method of detection such as a conjugate reactswith an exogenously added substrate. Various commercial kits areavailable, such as AlphaScreen® SureFire™ (PerkinElmer),ThermoScientific (Waltham, Mass., USA), Sigma Aldrich (St. Louis, Mo.,USA), ELISAOne (TGR BioSciences (South Australia, Australia) etc.).

As used herein, the term “binding”, such as in the context of thebinding of ligand to a receptor (e.g. GPCR), or such as an antibodybinding to an antigen, typically refers to an interaction or associationbetween a minimum of two entities, or molecular structures, such as areceptor-ligand interaction, or an antibody-antigen interaction. Thus a“receptor binding molecule” refers to a ligand or other moiety, such asa protein, that binds to, i.e. interacts with, a receptor.

For instance, binding affinity between the ligand (e.g., an apelinfusion protein) and the receptor (e.g., an APLNR or ligand bindingfragment of APLNR) typically corresponds to a K_(D) value of about 10⁻⁷M or less, such as about 10⁻⁸ M or less, such as about 10⁻⁹ M or less.Binding affinity can be determined by any one or more of severalmethods, such as by surface plasmon resonance (SPR) using a BIAcore 3000instrument. Accordingly, the ligand binds to the receptor with anaffinity corresponding to a K_(D) value that is at least ten-fold lower,such as at least 100 fold lower, for instance at least 1,000 fold lower,such as at least 10,000 fold lower, for instance at least 100,000 foldlower than its affinity for binding to a non-specific ligand (e.g., BSA,casein).

The term “K_(D)” (M), as used herein, refers to the dissociationequilibrium constant of a particular ligand-receptor interaction. Thereis an inverse relationship between K_(D) and binding affinity, thereforethe smaller the K_(D) value, the higher the affinity. Thus, the term“lower affinity” relates to a lower ability to form an interaction andtherefore a larger K_(D) value.

The term “k_(d)” (sec⁻¹ or 1/s), as used herein, refers to thedissociation rate constant of a particular ligand-receptor interaction.Said value is also referred to as the k_(off) value.

The term “k_(a)” (M⁻¹×sec⁻¹ or 1/M), as used herein, refers to theassociation rate constant of a particular ligand-receptor interaction.

The term “K_(A)” (M⁻¹ or 1/M), as used herein, refers to the associationequilibrium constant of a particular ligand-receptor interaction, or theassociation equilibrium constant of antibody-antigen interaction. Theassociation equilibrium constant is obtained by dividing the k_(a) bythe k_(d).

The term “EC₅₀” or “EC50”, as used herein, refers to the half maximaleffective concentration, which includes the concentration of a ligandthat induces a response, for example a cellular response, halfwaybetween the baseline and maximum after a specified exposure time. TheEC₅₀ essentially represents the concentration of a ligand where 50% ofits maximal effect is observed. Thus, with regard to cellular signaling,increased activity is observed with a decreased EC₅₀ value, i.e. halfmaximal effective concentration value (less ligand needed to effect agreater response).

In one embodiment, decreased binding refers to an increased EC₅₀ proteinconcentration, which enables half-maximal binding to the target receptoror receptor-expressing cells.

In some embodiments, decreased activity refers to an increased EC₅₀protein concentration, which enables half-maximal cellular activation ofthe target receptor or receptor-expressing cells.

The term “IC₅₀” or “IC50”, as used herein, refers to the half maximalinhibitory concentration of a cellular response. In other words, themeasure of the effectiveness of a particular moiety (e.g. protein,compound, or molecule) in inhibiting biological or biochemical function,wherein an assay quantitates the amount of such moiety needed to inhibita given biological process. Thus, with regard to cellular signaling, agreater inhibitory activity is observed with a decreased IC₅₀, orhalf-maximal inhibitory concentration, value.

In one embodiment, the apelin fusion protein is an agonist of the APLNRwith an EC50 of less than about 100 nM, or less than about 50 nM, orless than about 25 nM, or less than about 10 nM, or less than about 1nM, in an in vitro assay that measures activation of the APLNR. In oneembodiment, the apelin fusion protein comprises an Fc domain linked tothe N-terminus of an apelin peptide, and exhibits an EC50 of less thanabout 1 nM, or less than about 500 ρM.

Apelin Fusion Proteins of the Invention

Methods of making fusion proteins are known in the art. In one suchmethod, a DNA expression vector is engineered to contain anapelin-encoding nucleic acid sequence linked in-frame to an Fc-encodingnucleic acid sequence such that the DNA expression vector expresses onecontiguous fusion polypeptide. Apelin peptide may be linked to theC-terminus or to the N-terminus of the Fc-containing polypeptide. Apelinfusion proteins of the invention are expected to be more stable thanapelin peptides alone. Serum stable proteins include proteins thatconfer resistance to degradation or have a reduced clearance from thecirculation. Exemplary serum stable apelin fusion proteins of theinvention include SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 39, SEQ ID NO:40 and SEQ ID NO: 41.

In the context of constructing fusion proteins, the phrase “joinedin-frame” means that the components are linked together is such a waythat their complete translation, use or operation is possible and thusnot disrupted. For example, a fusion protein comprising at least twopolypeptides, may or may not have a linker or spacer sequence betweenthe polypeptides, and thus the polypeptides are joined in-frame as onecontinuous polypeptide with each polypeptide maintaining itsoperability. Two or more polypeptides linked or fused together in afusion protein are typically derived from two or more independentsources, and therefore a fusion protein comprises two or more linkedpolypeptides not normally found linked in nature. Furthermore, DNAencoding such fusion proteins may contain linker sequences that maintainoperable in-frame (e.g. triplet codon) translation of the transcribedmRNA molecules encoding such polypeptides.

The phrase “operably linked”, such as in the context of DNA expressionvector constructs, a control sequence, e.g., a promoter or operator, isappropriately placed at a position relative to a coding sequence suchthat the control sequence directs the production of a polypeptideencoded by the coding sequence.

The term “signal peptide” or “signal peptide sequence” is defined hereinas a peptide sequence usually present at the N-terminal end of newlysynthesized secretory or membrane polypeptides which directs thepolypeptide across or into a cell membrane of the cell (the plasmamembrane in prokaryotes and the endoplasmic reticulum membrane ineukaryotes). It is usually subsequently removed by enzyme cleavage. Insome embodiments, said signal peptide may be capable of directing thepolypeptide into a cell's secretory pathway. In some embodiments, thesignal peptide comprises the amino acid sequence from 1-29 of mouseROR1, GenBank Accession No. BAA75480 (SEQ ID NO: 10). In otherembodiments the signal peptide has at least about 96%, or at least about97%, or at least about 98%, or at least about 99% homology to the signalpeptide amino acid sequence shown in SEQ ID NO: 9. In still otherembodiments, the signal peptide is encoded by a nucleotide having atleast about 96%, or at least about 97%, or at least about 98%, or atleast about 99% homology to the signal peptide nucleic acid sequenceshown in SEQ ID NO: 9.

In some embodiments, the components or peptides of an Fc-fusion proteinare separated by a linker (or “spacer”) peptide. Such peptide linkersare well known in the art (e.g., polyglycine) and typically allow forproper folding of one or both of the components of the fusion protein.The linker provides a flexible junction region of the component of thefusion protein, allowing the two ends of the molecule to moveindependently, and may play an important role in retaining each of thetwo moieties' appropriate functions. Therefore, the junction region actsin some cases as both a linker, which combines the two parts together,and as a spacer, which allows each of the two parts to form its ownbiological structure and not interfere with the other part. Furthermore,the junction region should create an epitope that will not be recognizedby the subject's immune system as foreign, in other words, will not beconsidered immunogenic. Linker selection may also have an effect onbinding activity of the fusion molecule. (See Huston, et al, 1988, PNAS,85:16: 5879-83; Robinson & Bates, 1998, PNAS 95(11):5929-34; Arai, etal. 2001, PEDS, 14(8):529-32; and Chen, X. et al., 2013, Advanced DrugDelivery Reviews 65:1357-1369.) In one embodiment, the apelin peptide isconnected to the C-terminus or to the N-terminus of the Fc-containingpolypeptide, or fragment thereof, via one or more peptide linkers.

The length of the linker chain may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 15 or more amino acid residues, but typically is between 5and 25 residues. Examples of linkers include polyGlycine linkers, suchas Gly-Gly, Gly-Gly-Gly (3Gly), 4Gly, 5Gly, 6Gly, 7Gly, 8Gly or 9Gly.Examples of linkers also include Gly-Ser peptide linkers such asSer-Gly, Gly-Ser, Gly-Gly-Ser, Ser-Gly-Gly, Gly-Gly-Gly-Ser,Ser-Gly-Gly-Gly, Gly-Gly-Gly-Gly-Ser, Ser-Gly-Gly-Gly-Gly,Gly-Gly-Gly-Gly-Gly-Ser, Ser-Gly-Gly-Gly-Gly-Gly,Gly-Gly-Gly-Gly-Gly-Gly-Ser, Ser-Gly-Gly-Gly-Gly-Gly-Gly,(Gly-Gly-Gly-Gly-Ser)n, and (Ser-Gly-Gly-Gly-Gly)n, wherein n=1 to 10.(Gly-Gly-Gly-Gly-Ser)n and (Ser-Gly-Gly-Gly-Gly)n are also known as(G4S)n and (S4G)n, respectively.

In one such embodiment of the invention, the apelin peptide is connectedto the C-terminus or to the N-terminus of the Fc-containing polypeptide,or fragment thereof, via one or more Gly-Ser peptide linkers.

In one embodiment, the peptide linker is (Gly-Gly-Gly-Gly-Ser)₁,(Gly-Gly-Gly-Gly-Ser)₂, (Gly-Gly-Gly-Gly-Ser)₃, or(Gly-Gly-Gly-Gly-Ser)₄. In one embodiment, the peptide linker comprises(Gly-Gly-Gly-Gly-Ser)₃ (SEQ ID NO: 11).

In some embodiments, the signal peptide is connected to the N-terminusof the Fc-fusion polypeptide via one or more peptide linkers or spacers.In some embodiments, the signal peptide is encoded upstream of theFc-fusion protein in an expression vector and a spacer is encodedin-frame between the signal peptide and N-terminus of the Fc-fusionprotein. In another embodiment, the peptide linker or spacer comprisesRSTGSPGSG (SEQ ID NO: 12).

Modified Apelin Fusion Polypeptides

In other embodiments, the sequence of any Fc-fusion protein of theinvention may be modified so that it does not comprise any acceptorsites for N-linked glycosylation. In still other embodiments, thesequence of any Fc-fusion protein of the invention may be modified toenhance or diminish antibody binding to the FcRn receptor, e.g., atacidic pH as compared to neutral pH. In other embodiments, the sequenceof any Fc-fusion protein of the invention may be modified to resistcleavage or degradation. As such, addition of one or more C-terminalamino acids to the apelin peptide of an apelin-Fc-fusion protein mayconfer increased stability, such as resistance to degradation. Withoutbeing bound by one theory, additional C-terminal amino acids mayeliminate susceptibility to cleavage sites within the peptide or fusionprotein. Stability may be conferred due to decreased or slowed clearancefrom the circulation (i.e. renal excretion or clearance). Suchmodifications to apelin peptides do not alter their ability to activatethe APLNR. Exemplary modified apelin peptides are included in Tables 3and 4, e.g. SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO:44. Exemplary apelin fusion proteins of the invention include SEQ ID NO:39, SEQ ID NO: 40 and SEQ ID NO: 41.

In general, proteins, including Fc-fusion proteins described herein maybe modified by inclusion of any suitable number of such modified aminoacids (including non-standard amino acids, discussed supra) and/orassociations with conjugated substituents. Suitability in this contextis generally determined by the ability to at least substantially retainthe Fc-fusion protein's associated selectivity and/or specificity, forexample binding to APLNR. The modified amino acid may, for instance, beselected from a glycosylated amino acid, a PEGylated amino acid, afarnesylated amino acid, a geranyl-geranylated amino acid, an acetylatedamino acid, a biotinylated amino acid, an amino acid conjugated to alipid moiety, or an amino acid conjugated to an organic derivatizingagent, or the like. The inclusion of one or more modified amino acidsmay be advantageous in, for example, further increasing polypeptideserum half-life, reducing polypeptide antigenicity, or increasingpolypeptide storage stability. Amino acid(s) are modified, for example,co-translationally or post-translationally during recombinant production(e.g., N-linked glycosylation at N—X—S/T motifs during expression inmammalian cells) or modified by synthetic means. Non-limiting examplesof a modified amino acid include a glycosylated amino acid, a sulfatedamino acid, a prenylated (e.g., farnesylated, geranyl-geranylated) aminoacid, an acetylated amino acid, an acylated amino acid, a fatty acylatedamino acid, a PEGylated amino acid, a biotinylated amino acid, acarboxylated amino acid, a phosphorylated amino acid, and the like.References adequate to guide one of skill in the modification of aminoacids are replete throughout the literature. Example protocols are foundin Walker, 1998, Protein Protocols On CD-Rom, Humana Press, Totowa, N.J.

Proteins, including Fc-fusion proteins of the invention may also bechemically modified by covalent conjugation to a polymer to, forinstance, further increase their circulating half-life. Exemplarypolymers, and methods to attach them to peptides, are illustrated in forinstance U.S. Pat. No. 4,766,106, U.S. Pat. No. 4,179,337, U.S. Pat. No.4,495,285 and U.S. Pat. No. 4,609,546. Additional illustrative polymersinclude polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., aPEG with a molecular weight of between about 1,000 and about 40,000,such as between about 2,000 and about 20,000, e.g., about 3,000-12,000g/mol). See, e.g., WO2012/125408, which describes a PEG-apelin-36, apolypeptide with prolonged inotropic effects in rats.

In one embodiment, proteins including Fc-fusion proteins comprising oneor more radiolabeled amino acids are provided. A radiolabeled antibodymay be used for both diagnostic and therapeutic purposes. In anotherembodiment, proteins, including Fc-fusion proteins of the presentinvention may be conjugated to a molecule which is a therapeutic agentor a detectable marker. In one embodiment, the therapeutic agent is acytotoxic agent, such as a radioisotope. Examples of radioisotopes forpolypeptides include, but are not limited to, ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y,⁹⁹Tc, and ¹²⁵I, ¹³¹I, ¹⁸⁶Re, and ²²⁵AC. Methods for preparingradiolabeled amino acids and related peptide derivatives are known inthe art (see for instance Junghans et al., in Cancer Chemotherapy andBiotherapy 655-686 (2nd edition, Chafner and Longo, eds., LippincottRaven (1996)) and U.S. Pat. No. 4,681,581, U.S. Pat. No. 4,735,210, U.S.Pat. No. 5,101,827, U.S. Pat. No. 5,102,990 (U.S. RE35,500), U.S. Pat.No. 5,648,471 and U.S. Pat. No. 5,697,902. For example, a radioisotopemay be conjugated by a chloramine T method. In further embodiments, adetectable marker may be a radiolabel, an enzyme, a chromophore, or afluorescent label.

Expression Systems

The invention provides an expression vector encoding a polypeptide, e.g.an apelin Fc-fusion protein of the invention. Such expression vectorsmay be used for recombinant production of polypeptides of the invention.

An expression vector in the context of the present invention may be anysuitable vector, including chromosomal, non-chromosomal, and syntheticnucleic acid vectors (a nucleic acid sequence comprising a suitable setof expression control elements). Examples of such vectors includederivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeastplasmids, vectors derived from combinations of plasmids and phage DNA,and viral nucleic acid (RNA or DNA) vectors. In one embodiment, anFc-fusion protein or polypeptide-encoding nucleic acid molecule iscomprised in a naked DNA or RNA vector, including, for example, a linearexpression element (as described in, for instance, Sykes and Johnston,Nat Biotech 12, 355-59 (1997)), a compacted nucleic acid vector (asdescribed in for instance U.S. Pat. No. 6,077,835 and/or WO 00/70087),or a plasmid vector such as pBR322, pUC 19/18, or pUC 118/119. Suchnucleic acid vectors and the usage thereof are well known in the art(see, for instance, U.S. Pat. No. 5,589,466 and U.S. Pat. No.5,973,972).

In another embodiment, the vector comprises a nucleic acid moleculeencoding a polypeptide of the invention, including an expression vectorcomprising the nucleic acid molecules described wherein the nucleic acidmolecule is operatively linked to an expression control sequence.

In one embodiment, the vector is suitable for expression of apolypeptide of the invention in a bacterial cell. Examples of suchvectors include expression vectors such as BlueScript (Stratagene), pINvectors (Van Heeke & Schuster, 1989, J Biol Chem 264, 5503-5509), pETvectors (Novagen, Madison, Wis.) and the like.

An expression vector may also or alternatively be a vector suitable forexpression in a yeast system. Any vector suitable for expression in ayeast system may be employed. Suitable vectors include, for example,vectors comprising constitutive or inducible promoters such as yeastalpha factor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al.,ed., 1987, Current Protocols in Molecular Biology, Greene Publishing andWiley InterScience New York; and Grant et al., 1987, Methods in Enzymol153, 516-544).

In other embodiments, the expression vector is suitable for expressionin baculovirus-infected insect cells. (Kost, T; and Condreay, J P, 1999,Current Opinion in Biotechnology 10 (5): 428-33.)

A vector comprising a nucleic acid molecule of the invention isprovided, wherein the nucleic acid molecule is operably linked to anexpression control sequence suitable for expression in a mammalian hostcell.

Expression control sequences are engineered to control and drive thetranscription of genes of interest, and subsequent expression ofproteins in various cell systems. Plasmids combine an expressible geneof interest with expression control sequences (i.e. expressioncassettes) that comprise desirable elements such as, for example,promoters, enhancers, selectable markers, operators, etc. In anexpression vector of the invention, Fc-fusion protein orantibody-encoding nucleic acid molecules may comprise or be associatedwith any suitable promoter, enhancer, selectable marker, operator,repressor protein, polyA termination sequences and otherexpression-facilitating elements.

“Promoter” as used herein indicates a DNA sequence sufficient to directtranscription of a DNA sequence to which it is operably linked, i.e.,linked in such a way as to permit transcription of the Fc-fusion proteinor antibody-encoding nucleotide sequence when the appropriate signalsare present. The expression of a Fc-fusion protein or antibody-encodingnucleotide sequence may be placed under control of any promoter orenhancer element known in the art. Examples of such elements includestrong expression promoters (e.g., human CMV IE promoter/enhancer or CMVmajor IE (CMV-MIE) promoter, as well as RSV, SV40 late promoter, SL3-3,MMTV, ubiquitin (Ubi), ubiquitin C (UbC), and HIV LTR promoters).

In some embodiments, the vector comprises a promoter selected from thegroup consisting of SV40, CMV, CMV-IE, CMV-MIE, RSV, SL3-3, MMTV, Ubi,UbC and HIV LTR.

Nucleic acid molecules of the invention may also be operably linked toan effective poly (A) termination sequence, an origin of replication forplasmid product in E. coli, an antibiotic resistance gene as selectablemarker, and/or a convenient cloning site (e.g., a polylinker). Nucleicacids may also comprise a regulatable inducible promoter (inducible,repressable, developmentally regulated) as opposed to a constitutivepromoter such as CMV IE (the skilled artisan will recognize that suchterms are actually descriptors of a degree of gene expression undercertain conditions).

Selectable markers are elements well-known in the art. Under theselective conditions, only cells that express the appropriate selectablemarker can survive. Commonly, selectable marker genes express proteins,usually enzymes, that confer resistance to various antibiotics in cellculture. In other selective conditions, cells that express a fluorescentprotein marker are made visible, and are thus selectable. Embodimentsinclude beta-lactamase (bla) (beta-lactam antibiotic resistance orampicillin resistance gene or ampR), bls (blasticidin resistance acetyltransferase gene), bsd (blasticidin-S deaminase resistance gene), bsr(blasticidin-S resistance gene), Sh ble (Zeocin® resistance gene),hygromycin phosphotransferase (hpt) (hygromycin resistance gene), tetM(tetracycline resistance gene or tetR), neomycin phosphotransferase II(npt) (neomycin resistance gene or neoR), kanR (kanamycin resistancegene), and pac (puromycin resistance gene).

In certain embodiments, the vector comprises one or more selectablemarker genes selected from the group consisting of bla, bls, BSD, bsr,Sh ble, hpt, tetR, tetM, npt, kanR and pac. In other embodiments, thevector comprises one or more selectable marker genes encoding greenfluorescent protein (GFP), enhanced green fluorescent protein (eGFP),cyano fluorescent protein (CFP), enhanced cyano fluorescent protein(eCFP), or yellow fluorescent protein (YFP).

For the purposes of this invention, gene expression in eukaryotic cellsmay be tightly regulated using a strong promoter that is controlled byan operator that is in turn regulated by a regulatory protein, which maybe a recombinant “regulatory fusion protein” (RFP). The RFP consistsessentially of a transcription blocking domain, and a ligand-bindingdomain that regulates its activity. Examples of such expression systemsare described in US20090162901A1, which is herein incorporated byreference in its entirety.

As used herein “operator” indicates a DNA sequence that is introduced inor near a gene in such a way that the gene may be regulated by thebinding of the RFP to the operator and, as a result, prevents or allowtranscription of the gene of interest, i.e. a nucleotide encoding apolypeptide of the invention. A number of operators in prokaryotic cellsand bacteriophage have been well characterized (Neidhardt, ed.,Escherichia coli and Salmonella; Cellular and Molecular Biology 2d. Vol2 ASM Press, Washington D.C. 1996). These include, but are not limitedto, the operator region of the LexA gene of E. coli, which binds theLexA peptide, and the lactose and tryptophan operators, which bind therepressor proteins encoded by the Lad and trpR genes of E. coli. Thesealso include the bacteriophage operators from the lambda P_(R) and thephage P22 ant/mnt genes, which bind the repressor proteins encoded bylambda cI and P22 arc. In some embodiments, when the transcriptionblocking domain of the RFP is a restriction enzyme, such as NotI, theoperator is the recognition sequence for that enzyme. One skilled in theart will recognize that the operator must be located adjacent to, or 3′to the promoter such that it is capable of controlling transcription bythe promoter. For example, U.S. Pat. No. 5,972,650, which isincorporated by reference herein, specifies that tetO sequences bewithin a specific distance from the TATA box. In specific embodiments,the operator is preferably placed immediately downstream of thepromoter. In other embodiments, the operator is placed within 10 basepairs of the promoter.

In certain embodiments, the operator is selected from the groupconsisting of tet operator (tetO), NotI recognition sequence (notfamiliar with this; I know NotI as a restriction enzyme), LexA operator,lactose operator, tryptophan operator and Arc operator (AO). In someembodiments, the repressor protein is selected from the group consistingof TetR, LexA, LacI, TrpR, Arc, LambdaC1 and GAL4. In other embodiments,the transcription blocking domain is derived from a eukaryotic repressorprotein, e.g. a repressor domain derived from GAL4. Bacterial operatorscan be employed in mammalian and other host cell systems (see, e.g., US20090162901A1, which is herein incorporated by reference).

In an exemplary cell expression system, cells are engineered to expressthe tetracycline repressor protein (TetR) and a protein of interest isplaced under transcriptional control of a promoter whose activity isregulated by TetR. Two tandem TetR operators (tetO) are placedimmediately downstream of a CMV-MIE promoter/enhancer in the vector.Transcription of the gene encoding the protein of interest directed bythe CMV-MIE promoter in such vector may be blocked by TetR in theabsence of tetracycline or some other suitable inducer (e.g.doxycycline). In the presence of an inducer, TetR protein is incapableof binding tetO, hence transcription then translation (expression) ofthe protein of interest occurs. (See, e.g., U.S. Pat. No. 7,435,553,which is herein incorporated by reference in its entirety.)

Another exemplary cell expression system includes regulatory fusionproteins such as TetR-ER_(LBD)T2 fusion protein, in which thetranscription blocking domain of the fusion protein is TetR and theligand-binding domain is the estrogen receptor ligand-binding domain(ER_(LBD)) with T2 mutations (ER_(LBD)T2; Feil et al., 1997, Biochem.Biophys. Res. Commun. 237:752-757). When tetO sequences were placeddownstream and proximal to the strong CMV-MIE promoter, transcription ofthe nucleotide sequence of interest from the CMV-MIE/tetO promoter wasblocked in the presence of tamoxifen and unblocked by removal oftamoxifen. In another example, use of the fusion proteinArc2-ER_(LBD)T2, a fusion protein consisting of a single chain dimerconsisting of two Arc proteins connected by a 15 amino acid linker andthe ER_(LBD)T2 (supra), involves an Arc operator (AO), more specificallytwo tandem arc operators immediately downstream of the CMV-MIEpromoter/enhancer. Cell lines may be regulated by Arc2-ER_(LBD)T2,wherein cells expressing the protein of interest are driven by aCMV-MIE/ArcO2 promoter and are inducible with the removal of tamoxifen.(See, e.g., US 20090162901A1, which is herein incorporated byreference.)

In some embodiments, a vector of the invention comprises a CMV-MIE/TetOor CMV-MIE/AO2 hybrid promoter.

The vectors of the invention may also employ Cre-lox recombination toolsto facilitate the integration of a gene of interest into a host genome.A Cre-lox strategy requires at least two components: 1) Cre recombinase,an enzyme that catalyzes recombination between two loxP sites; and 2)loxP sites (e.g. a specific 34-base pair by sequence consisting of an8-bp core sequence, where recombination takes place, and two flanking13-bp inverted repeats) or mutant lox sites. (See, e.g. Araki et al.,1995, PNAS 92:160-4; Nagy, A. et al., 2000, Genesis 26:99-109; Araki etal., 2002, Nuc Acids Res 30(19):e103; and US20100291626A1, all of whichare herein incorporated by reference). In another recombinationstrategy, yeast-derived FLP recombinase may be utilized with theconsensus sequence FRT (see also, e.g. Dymecki, S. M., 1996, PNAS93(12): 6191-6196).

In another aspect, a gene (i.e. a nucleotide sequence encoding arecombinant polypeptide of the invention) is inserted within anexpression-enhancing sequence of the expression cassette, and isoptionally operably linked to a promoter, wherein the promoter-linkedgene is flanked 5′ by a first recombinase recognition site and 3′ by asecond recombinase recognition site. Such recombinase recognition sitesallow Cre-mediated recombination in the host cell of the expressionsystem. In some instances, a second promoter-linked gene is downstream(3′) of the first gene and is flanked 3′ by the second recombinaserecognition site. In still other instances, a second promoter-linkedgene is flanked 5′ by the second recombinase site, and flanked 3′ by athird recombinase recognition site. In some embodiments, the recombinaserecognition sites are selected from a loxP site, a lox511 site, alox2272 site, and a FRT site. In other embodiments, the recombinaserecognition sites are different. In a further embodiment, the host cellcomprises a gene capable of expressing a Cre recombinase.

In some embodiments, the vector further comprises anX-box-binding-protein 1 (mXBP1) gene capable of enhancing proteinproduction/protein secretion through control of the expression of genesinvolved in protein folding in the endoplasmic reticulum (ER). (See,e.g. Ron D, and Walter P., 2007, Nat Rev Mol Cell Biol. 8:519-529).

The term “cell” includes any cell that is suitable for expressing arecombinant nucleic acid sequence. Cells include those of prokaryotesand eukaryotes (single-cell or multiple-cell), bacterial cells (e.g.,strains of E. coli, Bacillus spp., Streptomyces spp., etc.),mycobacteria cells, fungal cells, yeast cells (e.g. S. cerevisiae, S.pombe, P. partoris, P. methanolica, etc.), plant cells, insect cells(e.g. SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni,etc.), non-human animal cells, mammalian cells, human cells, or cellfusions such as, for example, hybridomas or quadromas. In certainembodiments, the cell is a human, monkey, ape, hamster, rat or mousecell. In other embodiments, the cell is eukaryotic and is selected fromthe following cells: CHO (e.g. CHO K1, DXB-11 CHO, Veggie-CHO), COS(e.g. COS-7), retinal cells, Vero, CV1, kidney (e.g. HEK293, 293 EBNA,MSR 293, MDCK, HaK, BHK21), HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065,HL-60, Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127cell, SP2/0, NS-0, MMT cell, tumor cell, and a cell line derived from anaforementioned cell. In some embodiments, the cell comprises one or moreviral genes, e.g. a retinal cell that expresses a viral gene (e.g. aPER.C6® cell).

In some embodiments, the cell is a CHO cell. In other embodiments, thecell is a CHO K1 cell.

For example, in one embodiment, the present invention provides a hostcell comprising a nucleic acid stably integrated into the cellulargenome that comprises a nucleotide sequence coding for expression of arecombinant polypeptide of the present invention. In another embodiment,the present invention provides a cell comprising a non-integrated (i.e.,episomal) nucleic acid, such as a plasmid, cosmid, phagemid, or linearexpression element, which comprises a sequence coding for expression ofa recombinant polypeptide of the invention. In other embodiments, thepresent invention provides a cell line produced by stably transfecting ahost cell with a plasmid comprising an expression vector of theinvention.

In a further aspect, the invention relates to a method for producing anFc-fusion protein of the invention, said method comprising the steps ofa) culturing a host cell of the invention as described herein above, andb) purifying the Fc-fusion protein (supra) from the culture media.

Therapeutic and Diagnostic Uses of the Invention

In an even further aspect, the invention relates to a compositioncomprising an apelin fusion polypeptide or protein as defined herein.

The compositions may be formulated with pharmaceutically acceptablecarriers or diluents as well as known adjuvants and excipients inaccordance with conventional techniques such as those disclosed inRemington: The Science and Practice of Pharmacy, 19th Edition, Gennaro,Ed., Mack Publishing Co., Easton, Pa., 1995, and using trial and errorexperimentation.

The pharmaceutically acceptable carriers or diluents as well as anyother known adjuvants and excipients should be suitable for the chosenapelin fusion or apelin Fc-fusion protein of the present invention andthe chosen mode of administration. The actual dosage levels of theactive ingredients in the pharmaceutical compositions of the presentinvention may be varied so as to obtain an amount of the activeingredient which is effective to achieve the appropriate stability ofdrug substance, desired therapeutic response for a particular patient,composition, and mode of administration. The selected dosage level willdepend upon a variety of pharmacokinetic factors.

The pharmaceutical composition may be administered by any suitable routeand mode. Suitable routes of administering an apelin fusion protein ofthe present invention in vivo are well known in the art and may beselected by those of ordinary skill in the art. (Daugherty, A L, andMsrny, R J, 2006, Adv Drug Delivery Rev, 58(5-6): 686-706).

Apelin fusion proteins are agents administered for the management ofcardiovascular conditions, such as inotropic agents, specificallypositive inotropic agents. Without being bound to a particular theory,positive inotropic agents increase myocardial contractility, and areused to support cardiac function in conditions such as congestive heartfailure, myocardial infarction, cardiomyopathy, and others. (See Dai, etal., 2006, Eur J Pharmacol 553(1-3): 222-228; Maguire, et al,Hypertension. 2009; 54:598-604; and Berry, M., et al., 2004 Circulation,110:11187-11193.) Apelin-induced vasodilation may be protective inischemia-reperfusion injury. Promotion of angiogenesis and induction oflarger nonleaky vessels by apelin peptides may contribute to functionalrecovery from ischemia. (Eyries M, et al., 2008, Circ Res 103:432-440;Kidoya H, et al., 2010, Blood 115:3166-3174).

Apelin receptor agonists are considered pro-angiogenic agents which areadministered to increase cardiac output, improve cardiac function,stabilize cardiac function, limit a decrease in cardiac function, orpromote new blood vessel growth in an ischemic or damaged area of theheart or other tissue. Thus, apelin receptor agonists of the inventionare useful to promote angiogenesis and therefore treat ischemia, restorebloodflow to ischemic organs and tissues, for example to treat limbischemia, peripheral ischemia, renal ischemia, ocular ischemia, cerebralischemia, or any ischemic disease.

Apelin fusion proteins of the invention are agents administered toincrease blood flow, or increase heart contractility, such as to treator alleviate ischemia and heart failure.

Apelin fusion proteins are agents administered to treat or alleviateischemia and reperfusion injury, such as to limit ischemia/reperfusion(I/R) injury or delay the onset of necrosis of the heart tissue, or toprovide preventive treatment, for example, to protect the heart fromischemia/reperfusion (I/R) injury, improve cardiac function, or limitthe development myocardial infarction.

Apelin fusion proteins are agents administered for the management ofmetabolic conditions related to diabetes and obesity. Apelin improvesglucose tolerance and enhances glucose utilization, by muscle tissue, inobese insulin-resistant mice (Dray et al., 2008, Cell Metab 8:437-445).Apelin KO mice have diminished insulin sensitivity (Yue at al., 2010, AmJ Physiol Endocrinol Metab 298:E59-E67). As such, Apelin fusion proteinsare agents administered to improve glucose-tolerance in the treatment ofinsulin-resistant diabetes.

Changes in muscle apelin mRNA levels are also correlative withwhole-body insulin sensitivity improvements (Besse-Patin, A. et al.,2013 Aug. 27, Int J Obes (Lond). doi: 10.1038/ijo.2013.158, Epub aheadof print). Due to such metabolic improvements in muscle tissue, andapelin-induced vasodilation, agonistic apelin fusion proteins may alsobe administered to stimulate muscle growth and endurance.

It has been shown that primary HIV-1 isolates can also use APLNR as acoreceptor and synthetic apelin peptides inhibited HIV-1 entry intoCD4-APLNR-expressing cells (Cayabyab, M., et al., 2000, J. Virol., 74:11972-11976). Apelin fusion proteins are administered to treat HIVinfection.

Apelin-neuroprotection is also seen where apelin peptides act throughsignaling pathways to promote neuronal survival (Cheng, B, et al., 2012,Peptides, 37(1):171-3). Apelin fusion proteins are administered topromote or increase survival of neurons.

An apelin receptor agonist is also described as a hot flash suppressant.(See WO2012/133825, published Oct. 4, 2012.) Apelin fusion proteins ofthe invention may also be administered to treat, improve or suppress hotflash symptoms in a subject.

Apelin peptide may promote obesity through adipose tissue expansion.Apelin is induced by hypoxia and drives angiogenesis within the hypoxicinterior of expanding adipose tissue. (Kunduzova O, et al., 2008, FASEBJ, 22:4146-4153). Some apelin fusion proteins however are antagonists ofthe APLNR that act as inhibiting agents of this mechanism, in atissue-specific manner, to promote weight loss or treat obesity.Therefore, apelin fusion proteins are blocking agents administered totreat obesity and to promote weight loss.

Pathological angiogenesis, involved in promoting tumor growth orneovascularization in the retina may be responsive to apelin or APLNRantagonist. (Kojima, Y. and Quertermous, T., 2008, Arterioscler ThrombVasc Biol, 28:1687-1688; Rayalam, S. et al. 2011, Recent Pat AnticancerDrug Discov 6(3):367-72). As such, apelin fusion proteins are inhibitingagents administered to slow tumor growth or metastasis, or to treatcancer and metastatic disease. Apelin fusion proteins are alsoadministered to treat retinopathy.

APLNR antagonists may also reduce angiogenesis and improve function,such as in fibrotic tissues, by ameliorating the effects of anoveractive apelin system caused by a pathogenic disease (Principe, etal., 2008; Reichenbach, et al., 2012, JPET 340(3):629-637). Withoutbeing bound by any one theory, blocking the apelin system may slow theformation of excess fibrous connective tissue in an organ or tissue in areparative or reactive process, such as in a pathological condition likecirrhosis. As such, apelin fusion proteins may be used as inhibitingagents administered to slow or prevent the progression of fibrosis, orto treat fibrosis.

In some embodiments, Fc-fusion proteins of the invention provide amethod for the treatment of a disease or condition, the methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of an apelin fusion protein sufficient to treat thedisease or condition.

In one embodiment, provided herein is a method for treatment of adisease or condition related to apelin in a subject in need thereof, themethod comprising administering to the subject a therapeuticallyeffective amount of an apelin fusion protein.

In some embodiments, the apelin fusion protein comprises a polypeptidecomprising an apelin peptide fused to an Fc domain, or a fragmentthereof.

Diseases or conditions are selected from the group consisting ofcardiovascular disease, acute decompensated heart failure, congestiveheart failure, myocardial infarction, cardiomyopathy, ischemia,ischemia/reperfusion injury, pulmonary hypertension, diabetes, obesity,cancer, metastatic disease, fluid homeostasis, pathologicalangiogenesis, retinopathy, and HIV infection.

In some embodiments, the apelin fusion protein is an APLNR agonistuseful for treating a disease or condition selected from the groupconsisting of cardiovascular disease, acute decompensated heart failure,congestive heart failure, myocardial infarction, cardiomyopathy,ischemia, ischemia/reperfusion injury, pulmonary hypertension, diabetes,hot flash symptoms, fluid homeostasis, and HIV infection. In anotherembodiment, the apelin fusion protein is an APLNR agonist that promotesneuronal cell survival. In another embodiment, the apelin fusion proteinis an APLNR agonist that decreases sensitivity to insulin.

In some embodiments, the apelin fusion protein is an APLNR antagonistuseful for treating a disease or condition selected from the groupconsisting of obesity, cancer, metastatic disease, retinopathy,fibrosis, and pathological angiogenesis. In one embodiment, the apelinfusion protein is an APLNR antagonist that promotes weight loss. In oneembodiment, the apelin fusion protein is an APLNR antagonist thatdecreases pathological angiogenesis or neovascularization. In otherembodiments, the apelin fusion protein is an APLNR antagonist thatdecreases or inhibits tumor growth.

As used herein, a “therapeutically effective amount” of an Fc-fusionprotein means an amount sufficient to ameliorate, alleviate or partiallyarrest the clinical manifestations of a given disease and itscomplications in a therapeutic intervention comprising theadministration of said protein. An amount adequate to accomplish this isdefined as “therapeutically effective amount”. Effective amounts foreach purpose will depend on the severity of the disease or injury aswell as the weight and general state of the subject.

In the present context, the term “treatment” and “treating” means themanagement and care of a patient for the purpose of combating acondition, such as a disease or a disorder. The term is intended toinclude the full spectrum of treatments for a given condition from whichthe patient is suffering, such as administration of the activeingredient (Fc-fusion protein) to alleviate or relieve symptoms and/orcomplications, to delay the progression of the disease, disorder orcondition, and/or to remedy or eliminate the disease, disorder orcondition as well as to prevent the condition, wherein prevention is tobe understood as the management and care of a patient for the purpose ofstopping the disease progression, and includes the administration of theactive ingredients to prevent the onset of the symptoms orcomplications. Nonetheless, preventive, palliative, and therapeutic(curative) treatments are each aspects of the invention. The subject tobe treated is a mammal, in particular a human being.

In some embodiments, the treatment is maintenance treatment, recurrenceprevention or stabilization of the disease or condition.

The present invention includes compositions and therapeutic formulationscomprising any of the apelin fusion proteins described herein incombination with one or more additional therapeutically activecomponents, and methods of treatment comprising administering suchcombinations to subjects in need thereof.

Such additional therapeutically active components include VEGFinhibitors, blood pressure medication, calcium channel blockers,digitalis, anti-arrhythmics, ACE inhibitors, anti-coagulants,immunosuppressants, pain relievers, vasodilators, etc.

The apelin fusion proteins of the invention provide agents with improvedpharmacokinetic properties, such as circulating serum half-life andstability compared to apelin peptides that do not have an Fc domain orfragment of an Fc domain. In one embodiment, the apelin fusion proteinpost-injection serum level is increased or elevated for more than about1 hour, or more than about 2 hours, or more than about 3 hours, or morethan about 4 hours, or more than about 5 hours, or more than about 10hours, or more than about 24 hours. In other embodiments, the apelinfusion protein has a serum or plasma half-life of more than about 10minutes, or more than about 1 hour, or more than about 2, 3, 4, 5, 6, 7,8, 9, or more than about 10 hours, or more than about 24 hours.

Labeled apelin fusion proteins of the invention can be used fordiagnostic purposes to detect, diagnose, or monitor diseases ordisorders. The invention provides for the detection or diagnosis of adisease or disorder, comprising: (a) assaying the existence of apelinreceptor (APLNR) in cells or tissue samples of a subject using one ormore apelin fusion proteins that immunospecifically bind to the targetAPLNR; and (b) comparing the level of the APLNR with a control level,e.g. levels in normal tissue samples, whereby an increase in the assayedlevel of APLNR compared to the control level of APLNR is indicative ofthe disease or disorder, or indicative of the severity of the disease ordisorder.

Apelin fusion proteins of the invention can be used to assay APLNRlevels in a biological sample using immunohistochemical methodswell-known in the art. Other apelin-based methods useful for detectingAPLNR protein include immunoassays such as the enzyme linked immunoassay(ELISA) and the radioimmunoassay (RIA). Suitable apelin fusion proteinlabels may be used in such kits and methods, and labels known in the artinclude enzyme labels, such as alkaline phophatase and glucose oxidase;radioisotope labels, such as iodine (¹²⁵I, ¹³¹I) carbon (¹⁴C), sulfur(³⁵S), tritium (³H), indium (¹²¹In), and technetium (^(99m)Tc); andluminescent labels, such as luminol and luciferase; and fluorescentlabels, such as fluorescein and rhodamine.

Presence of labeled apelin fusion proteins may be detected in vivo fordiagnosis purposes. In one embodiment, diagnosis comprises: a)administering to a subject an effective amount of a labeled apelinfusion proteins; b) waiting for a time interval following administrationfor permitting labeled apelin fusion protein to concentrate at siteswhere APLNR may be detected and to allow for unbound labeled apelinfusion protein to be cleared to background level; c) determining abackground level; and d) detecting the labeled apelin fusion protein inthe subject, such that detection of labeled apelin fusion protein abovethe background level is indicative that the subject has increased APLNRprotein, or has the disease or disorder, or the increase APLNR proteinis indicative of the severity of the disease or disorder. In accordancewith such embodiment, the apelin fusion protein is labeled with animaging moiety suitable for detection using a particular imaging systemknown to those skilled in the art. Background levels may be determinedby various methods known in the art, including comparing the amount oflabeled apelin fusion protein detected to a standard value previouslydetermined for a particular imaging system. Methods and systems that maybe used in the diagnostic methods of the invention include, but are notlimited to, computed tomography (CT), whole body scan such as positronemission tomography (PET), magnetic resonance imaging (MRI), andsonography.

The invention also provides a pack or kit (e.g., a pharmaceutical packor kit) comprising one or more containers filled with at least oneactivating fusion protein of the invention. The kits of the inventionmay be used in any applicable method, including, for example,diagnostically. Optionally associated with such container(s) can be anotice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects (a) approval by the agency of manufacture, use orsale for human administration, (b) directions for use, or (c) bothapproval for manufacture and directions for use.

Ex Vivo and In Vivo Assays

Apelin Fc-fusion proteins of the invention maintain substantial activitywith respect to the APLNR while prolonging serum half-life. APLNR signaltransduction provides the nexus between apelin Fc-fusion proteins andthe known therapeutic and biological effects of apelin. Therefore, anydemonstration of an apelin Fc-fusion protein effect on APLNR activity invitro, ex vivo, or in vivo provides reasonable evidence of an in vivobiological or medical effect of the apelin Fc-fusion protein in apatient or animal. Among other studies, it has been demonstrated thatapelin/APLNR is an endogenous protective system against myocardialischemia/reperfusion (I/R) injury and the anti-apoptotic effects ofapelin/APLNR activation, specifically pERK, protects against such injury(Zeng, et al. 2009, Peptides, 30(6):1144-52, epub Feb. 24, 2009).

Agonists of APLNR, including endogenous apelin peptides, apelinanalogues, and modified apelin peptides, demonstrate therapeuticactivity in a number of in vivo assays (e.g. PEG-apelin-36, as inWO2012125408, and non-peptidic apelin agonists as in Iturrioz, X. et al.2010, FASEB J, 24(5):1506-17. Epub Dec. 29, 2009).

APLNR agonism has been demonstrated to result in increased heart rateand cardiac contractility (Ashley, E A, et. al. 2005, Cardiovasc Res.65(1):73-82). In addition, apelin peptide has been demonstrated to alterthe electrophysiology of cardiomyocytes. Whole-cell patch-clamptechniques were used to investigate the action potential (AP) and ioniccurrents in isolated rabbit left atrial (LA) myocytes before and afterthe administration of apelin (See, e.g., Farkasfalvi, K., et al., 2007,Biochem Biophys Res Commun. 357(4):889-95. Epub 2007 Apr. 12; and Cheng,C C, et al., 2013, Eur J Clin Invest. 43(1):34-40. Epub Oct. 28, 2012;which are both incorporated by reference herein). Isotropy induced byapelin agonism may also be assessed by measuring ECG parameters inisolated hearts from mice or rats using a Langendorf or Working HeartSystem. Such electrophysiological and in vivo techniques, such asmicro-ultrasound or echocardiography, are used to assess the therapeuticaction of the polypeptides of the invention.

The protective effects of apelin Fc-fusion polypeptides may be assessedfollowing myocardial ischemia/reperfusion (I/R) injury orhypoxia/re-oxygenation (H/R) in isolated rat or mouse hearts as in theLangendorf system (see e.g. Zeng, et al. 2009, Peptides, 30(6):1144-52,epub Feb. 24, 2009; Pisarenko, et al. 2010, Kardiologiia, 50(10):44-9;and Pisarenko, et al., 2013, J Pharmacol Pharmacother. “Effects ofstructural analogues of apelin-12 in acute myocardial infarction inrats”, epub before print).

Transient LAD ligation may also be performed, with apelin agonistadministered prior to reperfusion. (See Pisarenko, et al. 2011, Bull ExpBiol Med. 152(1):79-82; Li, L. et al, 2012, Am J Physiol Heart CircPhysiol, 303(5):H605-18, Epub Jun. 29, 2012; and Tao, J., et al, 2011,Am J Physiol Heart Circ Physiol, 301(4):H1471-86, Epub Jul. 29, 2011.)Following cardiac injury, microultrasound parameters may be used tomeasure cardiac function with respect to improvement, as well asassessment of infarct size.

The following examples are provided to describe to those of ordinaryskill in the art how to make and use methods and compositions of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure the accuracywith respect to numbers used (e.g. amounts, concentrations, temperature,etc.) but some experimental errors and deviations should be accountedfor.

EXAMPLES Example 1 Cloning of Expression Constructs

Synthetic gene fragments were used to generate N-terminal and C-terminalhFc fusions with apelin-13. DNA encoding the resulting fusions,hFc-Apelin13 (SEQ ID NO: 1) and Apelin13-hFc (SEQ ID NO: 3), wereinserted into expression vectors downstream of a CMV promoter, usingstandard molecular cloning techniques. CHO stable cell lines weregenerated and used for the production of fusion proteins, which werethen purified by affinity methods. N-terminal hFc-Apelin13 andC-terminal Apelin13-hFc fusion proteins migrate on SDS-PAGE gelsconsistent with their predicted mass. (See FIG. 3A.) Western blotanalysis, performed with the anti-apelin antibody (Abcam, #ab59469), wasused to confirm presence of apelin on hFc-Apelin13 and Apelin13-hFc.(See FIG. 3B.)

Example 2 Potency and Efficacy of Apelin Fc Fusion Proteins in acAMP-Reporter Assay

Modulation of intraceullular cAMP levels by unmodified apelin peptide(Bachem, #H-4568.0001) and apelin 13 fusion proteins of the inventionwas evaluated using a bioassay that was developed to detect theactivation of hAPLNR. A HEK293 cell line was transfected to stablyexpress the full-length human hAPLNR (amino acids 1-380 of accessionnumber NP_005152.1), along with a luciferase reporter [cAMP responseelement (CRE,4×)-luciferase]. The resulting cell line,HEK293/CRE-luc/hAPLNR, was maintained in DMEM containing 10% FBS, NEAA,pencillin/streptomycin, and 100 μg/mL hygromycin B. For the bioassay,HEK293/CRE-luc/hAPLNR cells were seeded onto 96-well assay plates at20,000 cells/well in 80 μL of OPTIMEM supplemented with 0.1% FBS andpenicillin/streptomycin/L-glutamine and incubated for 16 hours at 37° C.in 5% CO₂. The next morning, to measure inhibition of forskolin-inducedcAMP production via hAPLNR activation, unmodified apelin peptide andapelin 13 fusion proteins were serially diluted (1:3) then mixed withforskolin (Sigma, # F6886) in assay buffer (5 μM final forskolinconcentration), and added to the cells. After 5 hours of incubation at37° C. in 5% CO₂, luminescence was measured following the addition ofOne Glo reagent (Promega, #E6051) using a Victor X instrument (PerkinElmer). The data were fit by nonlinear regression to a 4-parameterlogistic equation with Prism 5 software (Graph Pad).

The hFc-Apelin13 fusion protein promoted inhibition of cAMP release fromforskolin-stimulated HEK293/CRE-luc/hAPLNR cells with an EC₅₀ value of174 ρM and Apelin13-hFc activated with an EC₅₀ value of 22.1 nM. In thisassay, apelin-13 activated with an EC₅₀ value of 36.5 ρM. (See FIG. 4.)

Example 3 Potency and Efficacy of Fc Fusion Proteins in a β-ArrestinAssay

The DiscoverX PathHunter® platform is based on the recruitment ofβ-arrestin to GPCRs in response to treatment with a relevant ligand. Inthis assay format, β-arrestin is fused to an N-terminal deletion mutantof β-galactosidase (β-gal) and stably-expressed in the cells whereas theGPCR is fused to a smaller (42 amino acids), weakly complementing β-galfragment. Ligand stimulation of the GPCR in this assay results in therecruitment of β-arrestin to the GPCR, forcing the complementation ofthe two β-gal fragments and resulting in the formation of a functionalenzyme that converts substrate to detectable signal (DiscoverXCorporation, Fremont, Calif., USA).

For the assay, CHO-K1/hAPLNR DiscoverX cells were plated at 10,000 cellsper well in assay media (DiscoverX Corporation; #93-0250E2) andincubated for 48 hours at 37° C. in 5% CO₂. Cells were then treated witha 1:10 serial dilution of either unmodified apelin-13 peptide or theapelin-13 fusion proteins. After 1.5 hours of incubation at 37° C.,detection reagents were added as per the manufacturer's specificationsand incubated for 1 hour at RT, followed by luminescence measurementusing a Victor instrument (Perkin-Elmer)

The hFc-Apelin13, apelin-13, and Apelin13-hFc proteins activatedCHO-K1/hAPLNR DiscoverX cells in a dose-dependent manner, with EC₅₀values of 992 ρM, 17.6 ρM, and 44.2 nM (extrapolated value),respectively (FIG. 5).

Example 4 Potency and Efficacy of Fc Fusion Proteins in a pERK Assay

To measure the effect of the apelin-13 fusion proteins of the inventionon the APLNR signaling pathway, an assay was used to quantify the amountof phosphorylated ERK1/2 (pERK1/2) and total ERK from an APLNRexpressing cell line. A Chinese hamster ovary (CHO) cell line wastransfected to stably express the full-length human APLNR (hAPLNR; aminoacids 1-380 of accession number NP_005152.1) under the control of adoxycycline-inducible CMV promoter. The resulting cell line, CHO/hAPLNRwas maintained in Ham's F12 media containing 10% FBS,penicillin/streptomycin, L-glutamine, and 250 ug/mL hygromycin B.

For the assay, CHO/hAPLNR cells were seeded onto 96 well assay plates at10,000 cells/well in 200 μL of Ham's F12 containing 10% FBS,L-glutamine, penicillin/streptomycin and incubated at 37° C. in 5% CO₂for 24 hours. The next day, to induce expression of the APLNR andprepare the cells for the pERK assay, the cells were first washed oncewith 250 μl of 1×PBS (Life Technologies; #20012-043), then serum-starvedin Ham's F12 containing 0.1% FBS, 1% BSA, L-glutamine,penicillin/streptomycin, 0.5 μg/mL doxycycline for 24 hours. On the dayof the assay, cells were treated with a 1:10 serial dilution of eitherunmodified apelin peptide or fusion proteins in Ham's F12 supplementedwith 1% BSA, penicillin/streptomycin, L-glutamine for 15 minutes at 37°C. in 5% CO₂. At the end of the incubation, cells were washed with 200μL of PBS and subsequently lysed with 100 uL of ELISAone Lysis Buffer(TGR BioSciences; #EBF001). Extracts were then analyzed forphosphorylated ERK (pERK1/2) and total ERK levels, as per themanufacturer's specifications (TGR Biosciences, #EKT001). Thefluorescence signals were then measured using a Spectramax plate reader(Molecular Devices). The ratio of measured pERK1/2 to measured total ERKwas calculated and the results were analyzed using GraphPad Prism.

In the pERK assay, hFc-Apelin13 and Apelin13-hFc increased the ratio ofpERK1/2 to total ERK1/2 in CHO/hAPLNR cells in a dose-dependent manner,with EC₅₀ values of 216 μM and 33 nM, respectively (FIG. 6).

Example 6 Pharmacokinetic Study to Evaluate Serum Stability of FcFusions

C57/B16 mice (n=3 per group) were dosed subcutaneously (s.c.) with hFc(2.5 mg/kg) or Apelin13-hFc (2.8 mg/kg) (FIG. 7A) and plasma wascollected at 1, 4, 24, and 48 hours. In a separate experiment,hFc-Apein13 was injected s.c. in C57/B16 mice (n=3 per group) at 5 mg/kgand serum was collected in 0, 1, 2, 4, 5, 6, 24 hours and 2, 3, 7, 14,21 days (FIG. 7B).

To evaluate serum/plasma levels of the administered proteins, 96-wellELISA plates were coated for 18 hrs at 4° C. with a 100 μL/well of goatanti-human IgG antibody (Jackson ImmunoLab; 109-005-098) at aconcentration of 1 μg/mL in PBS. The plates were subsequently blockedfor 1 hour at room temperature (RT) with 300 μL/well of 1× milkdiluent/blocking solution (KPL; #100108). Dilutions of hFc (for standardcurve) and serum samples in 100 μL of diluent were then added to theplate. After incubating for 2 hours at RT, the wells were then washed,and plate-bound human Fc was detected by addition of a horse-radishperoxidase conjugated anti-human IgG antibody (Jackson ImmuLab;#109-035-098) to the plate for 7 minutes at RT. Samples were developedfor 7 minutes with a TMB solution (MP Biomedical; #152346) to produce acolorimetric reaction and then neutralized with 100 μL/well of 2.0NH₂SO₄ (Mallinckrodt; #H381-05) before measuring absorbance at 450 nmwavelength on a Spectramax plate reader (Molecular Devices). Data wereanalyzed using SoftMax software to determine concentrations of thesamples in serum.

Apelin13-hFc serum levels reached a maximum of 10 μg/mL (380 nM) at ˜4hours and remained comparable to those of hFc after 48 hrs (FIG. 7A).The hFc-Apelin13 serum levels reached a maximum of 3 μg/mL (100 nM) at24 hours and gradually decreased to 1 μg/mL (38 nM) at day 14 (FIG. 7B).

Example 7 Potency and Efficacy of Apelin Peptides in a CRE Assay

Apelin-13 having an Fc tethered to its N-terminus (hFc-Apelin13)displays better potency than Apelin-13 having Fc tethered to itsC-terminus (Apelin-hFc), as seen in above Examples 2 through 6. ModifiedApelin-13 peptides, such as Apelin-13 peptides having one or more aminoacid(s) deleted from or added to the N-terminus or C-terminus, weretested for their relative potencies with respect to APLNR activation.

Modulation of cAMP levels by unmodified apelin-13 peptide (Bachem,#H-4568.0001) and modified apelin peptides of the invention wereevaluated using a bioassay that was developed to detect the activationof hAPLNR, according to the method of Example 2 (supra). The resultswere analyzed using nonlinear regression (4-parameter logistics) withPrism 5 software (GraphPad).

As shown in Table 3, apelin-13 can tolerate deletions of amino acidsfrom both the N-terminus and C-terminus while still retaining fullefficacy, and displaying different degrees of reduced potency comparedto apelin-13. Furthermore, apelin-13 can tolerate the addition of aminoacid residues to its C-terminus, such as five glycine residues, andstill retain full efficacy but with reduced potency, relative toapelin-13. It is envisioned that similar variations of Fc-apelin fusionproteins will maintain their efficacy.

TABLE 3 Apelin Peptides and Derivatives Maintain  Efficacy in CRE AssayApelin  Amino Acid Peptide Sequence EC₅₀ (M) Apelin-13  QRPRLSHKGPMPF1.403e−013 (SEQ ID NO: 6) Apelin-F13A  QRPRLSHKGPMPA 1.027e−010(SEQ ID NO: 29) Apelin65-76  QRPRLSHKGPMP 5.713e−011 (SEQ ID NO: 30)Apelin65-75  QRPRLSHKGPM 3.604e−012 (SEQ ID NO: 31) Apelin-12 RPRLSHKGPMPF 8.704e−013 (SEQ ID NO: 32) Apelin-11  PRLSHKGPMPF4.379e−010 (SEQ ID NO: 33) Apelin66-76  RPRLSHKGPMP 5.194e−012(SEQ ID NO: 34) Apelin67-76  PRLSHKGPMP 1.137e−013 (SEQ ID NO: 35)Apelin66-75  RPRLSHKGPM 2.174e−012 (SEQ ID NO: 36) Apelin67-75 PRLSHKGPM 3.738e−007 (SEQ ID NO: 37) Apelin-13 + 5G  QRPRLSHKGPMPF1.469e−010 (SEQ ID NO: 38) GGGGG

Example 8 Potency and Efficacy of Modified Apelin Fusion Proteins in aCRE Assay

Various apelin-Fc fusion proteins were made analogously to Example 1,except having modified apelin peptides, such as SEQ ID NO: 42, SEQ IDNO: 43 and SEQ ID NO:44, fused to the hFc. Such hFc-Apelin13 fusionproteins have an additional C-terminal amino acid at the C-terminus ofthe apelin peptide component. Modulation of cAMP levels by apelin-13peptide compared to these modified Apelin-13 peptides with hFc tetheredto its N-terminus (hFc-Apelin13+) were evaluated using the CRE bioassayanalogously to the methods of Example 2 and Example 7 (supra). Theresults were analyzed using nonlinear regression (4-parameter logistics)with Prism 5 software (GraphPad).

As shown in Table 4, modified apelin fusion proteins (having an Fc atthe N-terminus and additional amino acid at the C-terminus of the apelinpeptide component) exhibit activity at the APLNR similar to that ofunmodified apelin-13. The hFc-Apelin13 fusion protein having anadditional arginine at the C-terminus activated HEK293/CRE-luc/hAPLNRcells with an EC₅₀ value of 60 ρM. The hFc-Apelin13 fusion proteinhaving an additional serine at the C-terminus, and the hFc-Apelin13fusion protein having an additional histidine at the C-terminus, eachactivated APLNR with an EC₅₀ value of 96 ρM and 203 ρM, respectively. Inthis assay, apelin-13 activated with an EC₅₀ value of 56 ρM.

TABLE 4 Modified Apelin Fusion Proteins Maintain Efficacy in CRE AssayFusion SEQ ID NO: Protein tested (apelin peptide SEQ ID NO:) EC₅₀ (pM)apelin-13 — (SEQ ID NO: 6) 56 hFc-Apelin13-R SEQ ID NO: 39 (SEQ ID NO:42) 60 hFc-Apelin13-S SEQ ID NO: 40 (SEQ ID NO: 43) 96 hFc-Apelin13-HSEQ ID NO: 41 (SEQ ID NO: 44) 203

Example 9 Cardiovascular Evaluation of Apelin Fc Fusions

The effects of apelin Fc-fusion proteins of the invention are assessedby electrocardiography in anesthetized mice and rats, particularlyeffects on RR interval (index of heart rate) as well as QT interval asan index of ion channel activity.

The effects of APLNR agonists on blood pressure, heart rate and activityby radio telemetry in mice and rats are assessed for apelin Fc-fusionproteins of the invention. This method involves the implantation of apressure transducer in the carotid artery to measure aorta bloodpressure, heart rate and activity, with continuous data monitoring.

Cardiac function is also assessed by determining changes in cardiaccontractility by APLNR agonists in vivo. One method is the use ofmicro-ultrasound, or echocardiography (ECG) in mice or rats. Uponapplication of apelin Fc-fusion proteins in mice or rats, alterations inleft ventricle cardiac function are monitored using measurement of leftventricle end diastolic and end systolic volumes (EDV and ESV). Otherparameters are also recorded, such as ventricle diameters and heartrate, in order to calculate cardiac output (CO), Ejection Fraction (EF),Stroke Volume (SV), Fractional Shortening (FS) from recorded images ofmicro-ultrasound scans.

Isotropy, either induced by APNLR agonists or blocked by antagonists, isalso assessed by measuring left ventricular pressure, and dP/dT (changein pressure over time), heart rate, and cardiac conductance by ECG inisolated hearts from mice or rats using a Langendorf or Working Heartsystem.

Myocardial ischemia/reperfusion: Effects of apelin Fc-fusionpolypeptides may be assessed following myocardial ischemia/reperfusion(I/R) injury or hypoxia/re-oxygenation (H/R) in isolated rat or mousehearts as in the Langendorf system (see e.g. Zeng, et al. 2009,Peptides, 30(6):1144-52, epub Feb. 24, 2009). Transient LAD ligation isperformed, with apelin Fc-fusion polypeptides administered prior toreperfusion. (See e.g. Pisarenko, et al. 2011, Bull Exp Biol Med.152(1):79-82.) Microultrasound measures of cardiac function (describedhereinabove) are applied to determine improvement in this context.Infarct size is assessed by standard histology techniques.

Relaxation of pre-constricted aortic rings is assessed as follows: Exvivo preparation of thoracic aorta from mouse or rat is suspended bytitanium wires to a force transducer. Rings are pre-constricted with avasoconstrictor (such as Phenylephrine, nor-epinephrine, ornor-adrenaline, endothelin or angiotensin II). An increase in diameterand a decrease in force as measured by the force transducer indicates anability to induce vasorelaxation. (See Iturrioz, X. et al. 2010, FASEBJ, 24(5):1506-17, Epub Dec. 29, 2009; and also the Multi Myograph systemas in Zhong, et al., 2007, Cardiovasc Res 74(3): 388-395.)

What is claimed:
 1. A polypeptide comprising the amino acid sequence ofSEQ ID NO:
 2. 2. A composition comprising the polypeptide of claim 1 andat least one pharmaceutically acceptable carrier or diluent.
 3. Anucleic acid molecule encoding the polypeptide of claim
 1. 4. Thenucleic acid molecule of claim 3 linked to a nucleotide sequenceencoding a signal peptide.
 5. The nucleic acid molecule of claim 4comprising the nucleotide sequence consisting of SEQ ID NO:
 25. 6. Avector comprising the nucleic acid molecule of claim
 5. 7. A cellcomprising the vector of claim
 6. 8. A cell comprising the nucleic acidmolecule of claim
 5. 9. The cell of claim 8, wherein the nucleic acid isstably integrated into the genome of the cell.
 10. The cell of claim 9,wherein the cell is a eukaryotic cell.
 11. The cell of claim 10, whereinthe cell is selected from the group consisting of CHO, COS, retinalcell, Vero, CV1, 293, MDCK, HaK, BHK, HeLa, HepG2, WI38, MRC 5, Colo 25,HB 8065, HL-60, Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, Lcell, C127 cell, SP2/0, NS-0, MMT cell, and tumor cell.
 12. The cell ofclaim 11, wherein the cell is an animal cell.
 13. The cell of claim 12,wherein the cell is a mammalian cell.
 14. The cell of claim 13, whereinthe cell is a CHO cell.
 15. The cell of claim 14, wherein the cell is aCHO-K1 cell.